U.S. patent number 6,995,015 [Application Number 09/570,217] was granted by the patent office on 2006-02-07 for pathogen-resistant grape plants.
This patent grant is currently assigned to University of Florida. Invention is credited to Dennis J. Gray, Zhijian Li, Jayasankar Subramanian.
United States Patent |
6,995,015 |
Subramanian , et
al. |
February 7, 2006 |
Pathogen-resistant grape plants
Abstract
The invention features a method of producing a grape somatic
embryo having resistance to a plant pathogen, the method including
the steps of (a) culturing a grape somatic embryo in a first liquid
culture medium that includes a plant growth regulator and a
phytotoxin from a plant pathogen; (b) exchanging the first liquid
culture medium for a second liquid culture medium not including the
phytotoxin; (c) recovering a living grape cell or grape cell
cluster from the second liquid culture, the living cell or cell
cluster being resistant to the pathogen; and (d) culturing the
grape cell or grape cell cluster in a third culture medium to
produce a grape somatic embryo.
Inventors: |
Subramanian; Jayasankar
(Tavares, FL), Li; Zhijian (Altamonde Springs, FL), Gray;
Dennis J. (Howey-in-the-Hills, FL) |
Assignee: |
University of Florida
(Gainesville, FL)
|
Family
ID: |
26832159 |
Appl.
No.: |
09/570,217 |
Filed: |
May 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60134275 |
May 14, 1999 |
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60148251 |
Aug 11, 1999 |
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Current U.S.
Class: |
435/430.1;
435/430; 800/276 |
Current CPC
Class: |
C07K
14/415 (20130101); C12N 15/8282 (20130101) |
Current International
Class: |
A01H
1/04 (20060101) |
Field of
Search: |
;435/418,430.1,430
;800/295,276,301 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 710 234 |
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Sep 1993 |
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FR |
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WO 93/11660 |
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Jun 1993 |
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WO |
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WO 93/23529 |
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Nov 1993 |
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WO |
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WO 94/13787 |
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Jun 1994 |
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WO |
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WO 95/19102 |
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Jul 1995 |
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WO |
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WO 97/49277 |
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Dec 1997 |
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WO |
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WO 99/11133 |
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Mar 1999 |
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WO |
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WO 99/59398 |
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Nov 1999 |
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WO |
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WO 00/70054 |
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Nov 2000 |
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WO |
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|
Primary Examiner: Kruse; David H
Attorney, Agent or Firm: Van Dyke; Timothy H. Beusse
Brownlee Wolter Mora & Maire
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit to U.S. Provisional Application No.
60/134,275, filed May 14, 1999, and 60/148,251, filed Aug. 11,
1999, each of which is hereby incorporated by reference.
Claims
What is claimed is:
1. A method of producing a grape somatic embryo having resistance
to a phytotoxin, said method comprising the steps of (a) culturing
a grape somatic embryo in a first liquid culture medium comprising
a plant growth regulator and said phytotoxin; (b) exchanging said
first liquid cluture medium for a second liquid culture medium not
comprising said phytotoxin; (c) recovering a living grape cell or
grape cell cluster from said second liquid culture, said living
cell or cell cluster being resistant to said phytotxin; and (d)
culturing said grape cell or grape cell cluster in a third culture
medium to produce a grape somatic embryo; wherein said phytotoxin
is from Elsinoe ampelina.
2. The method of claim 1, further comprising the step of (e)
transferring said grape somatic embryo to a germination medium to
grow a grape plant.
3. The method of claim 1, wherein said plant growth regulator of
step (a) is an auxin.
4. The method of claim 1, wherein steps (a) (d) are repeated in
sequence at least four times.
5. The method of claim 1, wherein said culture step (a) is for at
least five consecutive days.
Description
BACKGROUND OF THE INVENTION
This invention relates to plants having an increased level of
resistance to a pathogen and to methods for producing them.
Grapevines are a deciduous temperate fruit crop of ancient origin.
Grape production (65.times.10.sup.6 metric tons) exceeds that of
any other temperate fruit crop, and ranks third after Citrus and
banana production. In addition, due to its uses for fresh fruit,
juice, jelly, raisins, and wine, grapes surpass all other fruit
crops in value. Therefore, successful efforts to improve grapevines
are likely to have a major impact on commercial viticulture.
Current methods for improving grapevines are time-consuming and
labor intensive. For example, genetic improvement in grapes through
conventional breeding is severely limited by a number of factors
such as long pre-bearing age and varying ploidy levels. Cultivated
grapes are also highly heterozygous and do not generally breed true
from seeds. Moreover, grape breeding programs are expensive,
long-term projects. Although plant biotechnology is an attractive
alternative for genetic improvement in grapes (Kuksova et al.,
Plant Cell Tiss. Org. Cult. 49:17 27, 1997), in vitro genetic
manipulation can be addressed only if there is an effective
regeneration system. Accordingly, methods that reduce any of these
problems would represent a significant advancement in the art.
SUMMARY OF THE INVENTION
We have discovered methods for growing perennial grape embryogenic
cultures and for growing large quantities of somatic grape embryos
from such perennial embryogenic cultures in a relatively short
period using a liquid suspension culture. Several advantages are
provided by the present methods. These approaches, for example,
facilitate an extraordinarily high frequency of somatic embryo
formation and plant regeneration. Such frequencies have not been
previously reported for grapevine regeneration of any known
cultivar, and render the method useful for large-scale production
of clonal planting stock of grape plants. In addition, the methods
produce embryos free of such common abnormalities as fusion and
fasciations of somatic embryos. The methods of the invention also
result in enhanced embryogenic culture initiation frequency,
allowing for the production of highly embryogenic cultures that can
then be successfully carried through the subsequent stages of the
regeneration process to the whole plant level. Because of these
advantages, the methods of the invention are especially useful in
the application of biotechnology for the genetic improvement of
this crop.
Embryogenic cells that are resistant to a plant pathogen can be
selected in vitro using methods of the present invention. From
these cells, or from the culture medium, proteins whose expression
is upregulated in response to a pathogen (and the nucleic acid
molecules encoding them) are identified. The proteins and nucleic
acid molecules can then be used to produce pathogen-resistant
plants (i.e., a transgenic or non-transgenic plant expressing such
a protein) or to increase plant resistance to a pathogen (e.g., by
applying recombinant protein to the surface of a plant.
Accordingly, in a first aspect, the invention features a method of
producing a grape somatic embryo having resistance to a plant
pathogen, the method including the steps of (a) culturing a grape
somatic embryo in a first liquid culture medium that includes a
plant growth regulator and a phytotoxin from a culture of the plant
pathogen; (b) exchanging the first liquid culture medium for a
second liquid culture medium not including the phytotoxin; (c)
recovering a living grape cell or grape cell cluster from the
second liquid culture, the living cell or cell cluster being
resistant to the pathogen; and (d) culturing the grape cell or
grape cell cluster in a third culture medium to produce a grape
somatic embryo.
In a second aspect, the invention features a method for producing a
grape plant having resistance to a plant pathogen, the method
including the steps of (a) culturing a grape somatic embryo in a
first liquid culture medium that includes a plant growth regulator
and a phytotoxin from a culture of the plant pathogen; (b)
exchanging the first liquid culture medium for a second liquid
culture medium not including the phytotoxin; (c) recovering a
living grape cell or grape cell cluster from the second liquid
culture, the living cell or cell cluster being resistant to the
pathogen; (d) culturing the grape cell or grape cell cluster in a
third culture medium to produce a grape somatic embryo; and (e)
growing a plant from the grape somatic embryo.
In the methods of the first and second aspects, the phytotoxin may
be obtained, for example, from a bacterium or fungus. A preferred
plant growth regulator in step (a) is an auxin (e.g., 2,4-D, NAA,
NOA, or picloram). If desired, the second culture medium may also
include a plant growth regulator. In other preferred embodiments,
steps (a) (d) of the method are repeated at least two time, more
preferably at least three times, and most preferably at least four
or five times. The culture step (a) can be for a day or two, but is
preferably for at least four days, six days, or more. In preferred
embodiments, the culture step (a) is for at least nine or ten
days.
In a third aspect, the invention features a grape plant regenerated
from a cell or cell cluster that has been selected in the presence
of a phytotoxin from a plant pathogen, wherein the plant has an
increased level of resistance to the pathogen relative to a control
grape plant regenerated from a cell or cell cluster not selected in
the presence of the phytotoxin. The grape plant is preferably
expressing a protein at a level that is at least 25% greater than
the level of the protein in the control plant, wherein the protein
is selected from the group consisting of (i) a protein having a
molecular weight of about 8 kDa and including the polypeptide of
SEQ ID NO: 1; (ii) a protein having a molecular weight of about 22
kDa and including the polypeptide of SEQ ID NO: 2; (iii) a protein
having a molecular weight of about 22 kDa and including the
polypeptide of SEQ ID NO: 3; and (iv) a protein having a molecular
weight of about 33 kDa and including the polypeptide of SEQ ID NO:
4. More preferably, the grape plant is expressing a protein at a
level that is at least 50%, 100%, 200%, 300%, or even 500% greater
than the level of the protein in the control plant.
In a fourth aspect, the invention features a transgenic grape plant
containing a transgene encoding a polypeptide substantially
identical to the polypeptide having the amino acid sequence of SEQ
ID NO: 5, wherein the transgene is operably linked to a promoter.
In preferred embodiments, the nucleic acid molecule has the
nucleotide sequence of SEQ ID NO: 6, and the promoter is a
constitutive promoter, an inducible promoter, or a tissue-specific
promoter.
In a fifth aspect, the invention features a transgenic grape plant
containing a transgene encoding a PR-5 protein that confers on the
plant resistance to a pathogen, wherein the nucleic acid molecule
is operably linked to a constitutive promoter.
In a sixth aspect, the invention features a transgenic grape plant
containing a transgene encoding a thaumatin-like protein that
confers on the plant resistance to a pathogen, wherein the nucleic
acid molecule is operably linked to a constitutive promoter.
In a seventh aspect, the invention features a transgenic grape
plant containing a transgene encoding a lipid transfer protein that
confers on the plant resistance to a pathogen, wherein the nucleic
acid molecule is operably linked to a constitutive promoter. In a
preferred embodiment, the lipid transfer protein is substantially
identical to the amino acid of SEQ ID NO: 5.
In an eighth aspect, the invention features a plant component from
the plant of the third, fourth, fifth, sixth, or seventh
aspect.
In a ninth aspect, the invention features a method of selecting a
plant having pathogen resistance. The method includes determining
the levels of a protein in the plant, wherein the protein includes
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and wherein
the levels of the protein are directly proportional to the levels
of pathogen resistance in the plant. The pathogen may be, for
example, a bacterium or fungus.
In a tenth aspect, the invention features a substantially pure
polypeptide substantially identical to the sequence of SEQ ID NO:
5. Preferably, the polypeptide, when expressed in a grape plant,
confers on the plant increased pathogen resistance relative to the
plant not expressing the polypeptide.
In an eleventh aspect, the invention features a substantially pure
nucleic acid molecule encoding a polypeptide substantially
identical to the sequence of SEQ ID NO: 5. Preferably, the
polypeptide, when expressed in a grape plant, confers on the plant
increased pathogen resistance relative to the plant not expressing
the polypeptide. In one preferred embodiment, the nucleic acid
molecule has the sequence of SEQ ID NO: 6.
In a twelfth aspect, the invention features a method of identifying
a protein that confers on a plant pathogen resistance. The method
includes the steps of (a) culturing a grape somatic embryo in a
first liquid culture medium including a plant growth regulator and
a phytotoxin from a plant pathogen culture; (b) exchanging the
first liquid culture medium for a second liquid culture medium not
including the phytotoxin; (c) recovering a grape cell or grape cell
cluster from the second liquid culture; (d) culturing the grape
cell or grape cell cluster in a third culture medium to produce a
grape somatic embryo having resistance to the plant pathogen; (e)
recovering the grape somatic embryo having resistance to the plant
pathogen; and (f) identifying a protein that is expressed in the
grape somatic embryo and that is not expressed in a grape somatic
embryo not cultured in a culture medium including the phytotoxin
from the plant pathogen culture, wherein the identified protein is
a protein that confers on a plant pathogen resistance.
In a thirteenth aspect, the invention features another method for
identifying a protein that, when expressed in a grape plant,
confers on the plant pathogen resistance, the method including the
steps of (a) contacting an embryogenic cell, embryogenic culture,
or somatic embryo, with a plant pathogen; and (b) measuring the
level of expression of a protein, wherein an increased level of
expression of the protein by the embryogenic cell, embryogenic
culture, or somatic embryo, relative to an embryogenic cell,
embryogenic culture, or somatic embryo not contacted with the plant
pathogen, identifies the protein as one that, when expressed in a
plant, confers on the plant pathogen resistance. The level of
expression may be measured, for example, using SDS-PAGE, ELISA, or
Western Blot analysis. Protein level is preferably standardized in
comparison to total protein level.
In a fourteenth aspect, the invention features a method for
producing a plant having increased resistance to a plant pathogen,
the method including overexpressing a protein identified by the
method of twelfth aspect or the method of the thirteenth
aspect.
In a fifteenth aspect, the invention features a method for
decreasing pathogen-mediated damage to a plant, the method
including contacting the plant with a recombinant form of a protein
that exhibits increased level of expression following contact with
a pathogen.
In a sixteenth aspect, the invention features a method for
identifying a cell that is expressing a protein that confers
pathogen resistance. The method including the steps of (a)
contacting a cell with a phytotoxin from a pathogen culture; and
(b) monitoring disease resistance of the cell, wherein increased
pathogen resistance, relative to a control cell, identifies the
cell as a cell that is expressing a protein that confers on the
plant resistance to a pathogen.
In a seventeenth aspect, the invention features a substantially
pure polypeptide including the amino acid of SEQ ID NO: 1 and
having a molecular weight of about 8 kDa as determined by reducing
SDS-PAGE, wherein the polypeptide is expressed at an increased
level in a grape plant in response to contact with a filtrate of a
culture of Elsino.delta. ampelina.
In an eighteenth aspect, the invention features a substantially
pure polypeptide including the amino acid of SEQ ID NO: 3 and
having a molecular weight of about 22 kDa as determined by reducing
SDS-PAGE, wherein the polypeptide is expressed at an increased
level in a grape plant in response to contact with a filtrate of a
culture of Elsino.delta. ampelina.
In a nineteenth aspect, the invention features a DNA molecule that
hybridizes to the DNA of SEQ ID NO: 6.
In a twentieth aspect, the invention features a transgenic plant
containing a transgene that hybridizes to the DNA of SEQ ID NO: 6
under high stringency conditions.
In a twenty-first aspect, the invention features a regenerated
grape plant that is expressing a protein at a level that is at
least 25% greater than the level of the protein in a control grape
plant regenerated from a cell or cell cluster not selected in the
presence of a phytotoxin from a plant pathogen culture, wherein the
protein is selected from the group consisting of: (i) a protein
having a molecular weight of about 8 kDa and comprising the
polypeptide of SEQ ID NO: 1; (ii) a protein having a molecular
weight of about 22 kDa and comprising the polypeptide of SEQ ID NO:
2; (iii) a protein having a molecular weight of about 22 kDa and
comprising the polypeptide of SEQ ID NO: 3; and (iv) a protein
having a molecular weight of about 33 kDa and comprising the
polypeptide of SEQ ID NO: 4. Preferably the plant is expressing at
least two proteins (and more preferably at least three or even all
four) at levels that are at least 25% greater than the level of the
protein in a control grape plant.
In a twenty-second aspect, invention features a substantially pure
polypeptide comprising the amino acid sequence of SEQ ID NO: 4.
Terms used herein are defined as follows:
By "perennial grape embryogenic culture" is meant an embryogenic
culture in which embryogenic cells or cell masses have been
repeatedly selected, subcultured, and maintained as an in vitro
culture. Such perennial grape embryogenic cultures can be
maintained for at least half a year, preferably three years, and
most preferably four or more years.
By "embryogenic cell," "embryogenic cell mass," or "embryogenic
cultures" is meant a cell or collection of cells having the
inherent potential to develop into a somatic embryo and,
ultimately, into a plant. Typically such cells have large nuclei
and dense cytoplasm. Additionally, such cells are usually
totipotent in that they typically possess all of the genetic and
structural potential to ultimately become a whole plant.
By "increased level of embryogenesis" is meant a greater capacity
to produce an embryogenic cell or embryogenic cell mass in a
perennial grape embryogenic culture than the level of a control
non-perennial grape embryogenic culture. In general, such an
increased level of embryogenesis is at least 20%, preferably at
least 50%, more preferably at least 100% and most preferably at
least 250% or greater than the level of a control embryogenic
culture. The level of embryogenesis is measured using conventional
methods.
By "explant" is meant an organ, tissue, or cell derived from a
plant and cultured in vitro for the purpose of initiating a plant
cell culture or a plant tissue culture. For example, explant grape
tissue may be obtained from virtually any part of the plant
including, without limitation, anthers, ovaries, ovules, floral
tissue, vegetative tissue, tendrils, leaves, roots, nucellar
tissue, stems, seeds, protoplasts, pericycle, apical meristem
tissue, embryogenic tissue, somatic embryos, and zygotic
embryos.
By "plant growth regulator" is meant a compound that affects plant
cell growth and division. Preferred plant growth regulators include
natural or synthetic auxins or cytokinins. Exemplary auxins
include, but are not limited to, NOA, 2,4-D, NAA, IAA, dicamba, and
picloram. Exemplary cytokinins include, but are not limited to, BA
and zeatin.
By "somatic embryogenesis" is meant the process of initiation and
development of embryos in vitro from plant cells and tissues absent
sexual reproduction.
By "somatic embryo" is meant an embryo formed in vitro from somatic
cells or embryogenic cells by mitotic cell division.
By "mature somatic embryo" is meant a fully-developed embryo with
evidence of root and shoot apices and exhibiting a bipolar
structure. Preferred mature somatic embryos are those with
well-defined cotyledons.
By "plantlet" is meant a small germinating plant derived from a
somatic embryo.
By "regeneration" is meant the production of an organ, embryo, or
whole plant in plant tissue culture.
By "plant cell" is meant any cell containing a plastid. A plant
cell, as used herein, is obtained from, without limitation, seeds,
suspension cultures, embryos, meristematic regions, callus tissue,
protoplasts, leaves, roots, shoots, somatic and zygotic embryos, as
well as any part of a reproductive or vegetative tissue or
organ.
By "promoter" is meant a region of nucleic acid, upstream from a
translational start codon, which is involved in recognition and
binding of RNA polymerase and other proteins to initiate
transcription. A "plant promoter" is a promoter capable of
initiating transcription in a plant cell, and may or may not be
derived from a plant cell.
By "tissue-specific promoter" is meant that the expression from the
promoter is directed to a subset of the tissues of the plant. It
will be understood that not every cell in a given tissue needs to
be expressing from the promoter in order for the promoter to be
considered tissue-specific.
By "heterologous" is meant that the nucleic acid molecule
originates from a foreign source or, if from the same source, is
modified from its original form. Thus, a "heterologous promoter" is
a promoter not normally associated with the duplicated enhancer
domain of the present invention. Similarly, a heterologous nucleic
acid molecule that is modified from its original form or is from a
source different from the source from which the promoter to which
it is operably linked was derived.
The term "plant" includes any cell having a chloroplast, and can
include whole plants, plant organs (e.g., stems, leaves, roots,
etc.), seeds, and cells. The class of plants that can be used in
the method of the invention is generally as broad as the class of
higher plants amenable to transformation techniques, including both
monocots and dicots.
By "plant component" is meant a part, segment, or organ obtained
from an intact plant or plant cell. Exemplary plant components
include, without limitation, somatic embryos, leaves, fruits,
scions, cuttings, and rootstocks.
By "phytotoxin" is meant a substance that is capable of killing a
plant cell. Phytotoxins are preferably from a pathogen such as a
fungus or a bacterium. For use in the present invention, they may
be purified or unpurified. In one example, a phytotoxin is present
in a filtrate from a culture of a pathogen such as a bacterium or a
fungus. The identity of the phytotoxin (e.g., its chemical
structure) need not be known for use in the methods of the
invention.
By "pathogen" is meant an organism whose infection of viable plant
tissue elicits a disease response in the plant tissue. Such
pathogens include, without limitation, bacteria and fungi. Plant
diseases caused by these pathogens are described in Chapters 11 16
of Agrios, Plant Pathology, 3rd ed., Academic Press, Inc., New
York, 1988.
Examples of bacterial pathogens include, without limitation,
Agrobacterium vitis, Agrobacterium tumefaciens, Xylella fastidosa,
and Xanthomonas ampelina. Examples of fungal pathogens include,
without limitation, Uncinula necator, Plasmopara viticola, Botrytis
cinerea, Guignardia bidwellii, Phomophsis viticola, Elsinoe
ampelina, Eutypa lata, Armillaria mellea, and Verticllium
dahliae.
By "pathogen culture" is meant a culture in which a pathogen has
grown. A filtrate of the culture is preferably substantially free
of the pathogen.
By "increased level of resistance" is meant a greater level of
resistance or tolerance to a disease-causing pathogen or pest in a
resistant grapevine (or scion, rootstock, cell, or seed thereof)
than the level of resistance or tolerance or both relative to a
control plant (i.e., a grapevine that has not been subjected to in
vitro selection to any plant pathogen or toxin-containing filtrate
thereof). In preferred embodiments, the level of resistance in a
resistant plant of the invention is at least 5 10% (and preferably
at least 30% or 40%) greater than the resistance of a control
plant. In other preferred embodiments, the level of resistance to a
disease-causing pathogen is at least 50% greater, 60% greater, and
more preferably even more than 75% or even 90% greater than the
level of resistance of a control plant; with up to 100% above the
level of resistance as compared to the level of resistance of a
control plant being most preferred. The level of resistance or
tolerance is measured using conventional methods. For example, the
level of resistance to a pathogen may be determined by comparing
physical features and characteristics (for example, plant height
and weight, or by comparing disease symptoms, for example, delayed
lesion development, reduced lesion size, leaf wilting, shriveling,
and curling, decay of fruit clusters, water-soaked spots, leaf
scorching and marginal burning, and discoloration of cells) of
resistant grape plants with control grape plants. Quantitation can
be performed on the level of populations. For example, if 4 out of
40 control plants are resistant to a given pathogen, and 20 out of
40 plants of the invention are resistant to that pathogen, than the
latter plant is 20/4 or 500% more resistant to the pathogen.
By "transformed" is meant any cell which includes a nucleic acid
molecule (for example, a DNA sequence) which is inserted by
artifice into a cell and becomes part of the genome of the organism
(in either an integrated or extrachromosomal fashion for example, a
viral expression construct which includes a subgenomic promoter)
which develops from that cell. As used herein, the transformed
organisms or cells are generally transformed grapevines or
grapevine components and the nucleic acid molecule (for example, a
transgene) is inserted by artifice into the nuclear or plastidic
compartments of the plant cell.
By "transgene" is meant any piece of a nucleic acid molecule (for
example, DNA) which is inserted by artifice into a cell, and
becomes part of an organism (or a descendant thereof) by being
integrated into the genome or maintained extrachromosomally which
develops from that cell. Such a transgene may include a gene which
is partly or entirely heterologous (i.e., foreign) to the
transgenic organism, or may represent a gene homologous to an
endogenous gene of the organism.
By "transgenic plant" is meant a plant containing a transgene.
Those in the art will recognize that, once a transgenic plant has
been produced, it may be propagated sexually or asexually; if a
descendant contains a transgene, it is considered to be a
transgenic plant.
By "protein" is meant any combination of two or more
covalently-bonded amino acids, regardless of post-translational
modifications.
By "PR-5" is meant a protein that is substantially identical to
VVTL-1 (SP accession no. O04708) and, when overexpressed in a grape
plant, confers on the plant increased pathogen resistance.
Sequence identity is typically measured using sequence analysis
software with the default parameters specified therein (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University
Avenue, Madison, Wis. 53705). This software program matches similar
sequences by assigning degrees of homology to various
substitutions, deletions, and other modifications. Conservative
substitutions typically include substitutions within the following
groups: glycine, alanine, valine, isoleucine, leucine; aspartic
acid, glutamic acid, asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine.
By "high stringency conditions" is meant hybridization in
2.times.SSC at 40.degree. C. with a DNA probe length of at least 40
nucleotides. For other definitions of high stringency conditions,
see F. Ausubel et al., Current Protocols in Molecular Biology, pp.
6.3.1 6.3.6, John Wiley & Sons, New York, N.Y., 1994, hereby
incorporated by reference.
By "substantially pure polypeptide" is meant a polypeptide that has
been separated from the components that naturally accompany it.
Typically, the polypeptide is substantially pure when it is at
least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is at least 75%, more
preferably at least 90%, and most preferably at least 99%, by
weight, pure. A substantially pure polypeptide may be obtained, for
example, by extraction from a natural source, by expression of a
recombinant nucleic acid encoding the polypeptide, or by chemically
synthesizing the protein. Purity can be measured and further
enhanced by any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis.
Methods of measuring protein amounts are known in the art. Any of
these methods is useful for quantitating the level of total protein
or of a specific protein. For example, proteins can be separated by
polyacrylamide gel electrophoresis and individual proteins
quantitated using densitometry.
A polypeptide is substantially free of naturally associated
components when it is separated from those contaminants that
accompany it in its natural state. Thus, a polypeptide which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those which
naturally occur in eukaryotic organisms but are synthesized in E.
coli or other prokaryotes.
By "substantially pure nucleic acid" is meant nucleic acid that is
free of the genes which, in the naturally-occurring genome of the
organism from which the nucleic acid of the invention is derived,
flank the nucleic acid. The term therefore includes, for example, a
recombinant nucleic acid that is incorporated into a vector; into
an autonomously replicating plasmid or virus; into the genomic
nucleic acid of a prokaryote or a eukaryote cell; or that exists as
a separate molecule (e.g., a cDNA or a genomic or cDNA fragment
produced by PCR or restriction endonuclease digestion) independent
of other sequences. It also includes a recombinant nucleic acid
that is part of a hybrid gene encoding additional polypeptide
sequence.
The invention features plants that are resistant to pathogens and
method for their production. Other features and advantages of the
invention will be apparent from the following detailed description,
and from the claims.
DESCRIPTION OF THE DRAWINGS
FIG. 1A is a photograph of a plant culture plate showing the
embryogenic mass of `Chardonnay` obtained from a liquid culture
medium. This photograph was taken approximately ten weeks after the
initiation of a liquid cell culture.
FIG. 1B is a photograph of a plant culture plate showing
cotyledonary stage somatic embryos. This photograph was taken
approximately twelve weeks after the initiation of embryo
development.
FIG. 1C is a photograph of a plant culture plate showing mature
somatic embryos starting to precociously germinate in liquid
culture. Note the elongation of roots in many embryos. This
photograph was taken approximately twenty weeks after the
initiation of embryo development.
FIG. 1D is a photograph of a plant culture plate showing initial
stages of somatic embryo differentiation in a solid medium after
five weeks of culturing. These somatic embryos were obtained from
embryogenic cell masses that were cultured in a liquid medium (from
FIG. 1A).
FIG. 1E is a photograph of a plant culture plate showing the early
stages of somatic embryo development in a solid medium. Very early
somatic embryos are hyaline and start turning opaque after a few
days.
FIG. 1F is a photograph of a plant culture plate showing mature
somatic embryos germinating on a MS basal medium with 3%
sucrose.
FIG. 2 shows growth of resistant proembryogenic masses in
suspension culture after 4 cycles (10 days each) of in vitro
selection with medium containing 40% Elsinoe ampelina culture
filtrate.
FIGS. 3A and 3B are photographs showing inhibition of mycelial
growth in dual culture by in vitro selected line. In vitro selected
(left) and non-selected (right) PEMs from suspension were cultured
in semisolid medium for 6 weeks and a small mycelial plug (5 mm
diameter) was placed at the center after 6 weeks. Photograph taken
10 days after fungal inoculation. (FIG. 3A) Elsinoe ampelina, (FIG.
3B) Fusarium oxysporium isolated from watermelon.
FIG. 4 is a photograph showing mycelial growth inhibition of E.
ampelina in conditioned medium assay. Spent liquid medium, after
growing in vitro selected (RC1 and RC2) and non-selected (C) PEMs
in suspension, was solidified on glass cover slips, with potato
dextrose agar to give a final strength of 0.75N. Mycelial plug of
Elsinoe ampelina was inoculated and allowed to grow on the plates.
Photographed two weeks after fungal inoculation.
FIGS. 5A 5C are photographs of SDS-PAGE of extracellular proteins
precipitated from spent liquid medium after growing selected and
non-selected PEMs (FIG. 5A), somatic embryos (FIG. 5B), and
inter-cellular washing fluids (ICWF) (FIG. 5C) in suspension
culture. The gels were silver stained. Lanes (S) molecular weight
markers, (C) non-selected control, (1) resistant line RC1, (2)
resistant line RC2.
FIGS. 6A and 6B show chitinase activity in the extracellular
proteins precipitated from spent liquid medium after growing
selected and non-selected PEMs in suspension culture. Chitinase
activity was detected after native PAGE (FIG. 6A) or SDS-PAGE (FIG.
6B) using a glycol chitin assay. Lanes (c) non-selected control,
(s) Chitinase standard from Serratia marcescens (Sigma, St. Louis,
Mo.) (1) resistant line RC1 (2) resistant line RC2.
FIG. 7 shows mature somatic embryos of selected and non-selected
cultures growing in solid medium containing 40% (v/v) Elsinoe
ampelina culture filtrate. (Left) Somatic embryos of R1 in regular
embryogenesis medium, (Center) Somatic embryos of RC1 in
embryogenesis medium containing 40% (v/v) culture filtrate, (Right)
Somatic embryos of non-selected control in embryogenesis medium
containing 40% (v/v) culture filtrate.
FIG. 8 shows greenhouse grown, somatic embryo derived plants of
selected and non-selected cultures were sprayed with Elsinoe
ampelina spore suspension (1.times.10.sup.6 spores per ml). Plants
from non-selected somatic embryos exhibited anthracnose symptoms 4
days after inoculation (inset), while the in vitro selected plant
did not show any symptom.
FIG. 9 is a schematic illustration showing the nucleotide and amino
acid sequence of the 33 kDa protein.
FIGS. 10A 10C are a series of photographs showing immuno-detection
of a 22 kDa protein with PR-5 antiserum. Extracellular proteins
(ECPs) from PEMs and heart stage somatic embryos were separated by
SDS-PAGE on a 12% mini-gel and transferred to a ImmunBlot.TM.
membrane and detected with PR-5 antiserum from `Pinto bean`.
Proteins from PEMs are shown in FIG. 10A, heart stage somatic
embryos in FIG. 10B, and ICWF of regenerated plants in FIG. 10C.
Lanes: C--non-selected control, RC1--in vitro selected line 1, and
RC2--in vitro selected line 2.
FIG. 11 is a schematic illustration showing a comparison of the
amino terminal amino acid residues a 9 kDa protein from ECP of
heart stage somatic embryos. This protein had high homology with
several nsLTP: Vitis nsLTP P4 (SP accession no. P80274) (SEQ ID
NO:7), Vitis nsLTP (Salzman et al., Plant Physiol 117:465 472,
1998) (SEQ ID NO:8), Sorghum nsLTP (SP accession no. Q43194) (SEQ
ID NO:9), rice nsLTP (SP accession no. P23096) (SEQ ID NO:10). The
consensus amino acid sequence (SEQ ID NO:11) is also shown. X
denotes unidentified amino acid residue.
FIG. 12 is a schematic illustration showing a comparison of the
amino terminal amino acid residues a 22 kDa protein from ECP of
heart stage somatic embryos. This protein had high homology with
several TLPs: Vvtl 1 (SP accession no. O04708) (SEQ ID NO:12) of,
Tobacco TLP-E22 (accession no. P13046) (SEQ ID NO:13), Tobacco
TLP-E2 (accession no. P07052) (SEQ ID NO:14), Vvosm (accession no.
Y10992) (SEQ ID NO:15), and grape osmotin (GO; Salzman et al.,
supra) (SEQ ID NO:16). The consensus amino acid sequence (SEQ ID
NO:17) is also shown. X denotes unidentified amino acid
residue.
FIG. 13 is a schematic illustration showing a comparison of the
amino terminal amino acid residues of the two .about.22 kDa
proteins from regenerated, in vitro selected plants (SEQ ID NOs:18
and 19). X denotes unidentified amino acid residue.
DETAILED DESCRIPTION OF THE INVENTION
We have developed a method for growing perennial grape embryogenic
cultures that is useful for the regeneration of grape plants. The
unique germplasm resulting from our culture system has been
observed to produce grape plants with an enhanced ability to
recreate embryogenic cultures. Furthermore, we have developed a
process for growing large quantities of somatic grape embryos from
such perennial embryogenic cultures in a relatively short period
using a liquid suspension culture. The culture method is useful,
for example, for selecting somatic grape embryos capable of
surviving in the presence of a pathogen. We have discovered that
plants derived from these somatic embryos are also more resistant
to pathogens. The plants of the invention are likely to have
resistance to many pathogens. The "Compendium of Grape Diseases"
(APS Press (1988) R. C. Pearson & A. C. Goheen, Eds.; hereby
incorporated by reference) describes a wide variety of grape plant
diseases and the pathogens that cause them. These include, without
limitation, botrytis bunch rot and blight (Botrytis cinerea); black
rot (Guignardia bodwelli); phomopsis cane and leaf spot (Phomopsis
viticola); anthracnose (Elsinoe ampelina); bitter rot (Greeneria
uvicola); white rot (Coniella diplodiella); ripe rot
(Colletotrichum gloeosporioides); macrophoma rot (Botryosphavria
dothidea); angular leaf spot (Mycosphaerella nagulata); diplodia
cane dieback and bunch rot (Diplodia natelensis); rust (Physopella
ampelopsidis); leaf blight (Pseudocerospora vitis); leaf blotch
(Brioisia ampelaphaga); zonate leaf spot (Cristulariella moricola);
septoria leaf spot (Septoria spp.); eutypa dieback (Eutypa lata);
black dead arm (Botryosphaeria steuensil); phymatotrichum root rot
(Phymatotrichum omnivorum); verticillium wilt (Verticillium
dahliae); dematophora root rot; (Dematophora necatrix);
phytophthora crown and root rot (Phytophthora spp.); crown gall
(Agrobacterium spp.); bacteria blight (Xanthomas ampelina);
Pierce's disease (Xylella fastidiosa); flavescence doree; and bois
noir and vergilbungskrankheit, and other grapevine yellows.
The regeneration methods described herein have been used for the
successful regeneration by somatic embryogenesis of a variety of
grapevine rootstock and scion cultivars, including Autumn Seedless,
Blanc du Bois, Cabernet Franc, Cabernet Sauvignon, Chardonnay
(e.g., CH 01 and CH 02), Dolcetto, Merlot, Pinot Noir (e.g., PN and
PN Dijon), Semillon, White Riesling, Lambrusco, Stover, Thompson
Seedless, Niagrara Seedless, Seval Blanc, Zinfindel, Vitis
rupestris St. George, Vitis rotundifolia Carlos, Vitis rotundifolia
Dixie, Vitis rotundifolia Fry, and Vitis rotundifolia Welder. The
methods of the invention are generally applicable for a variety of
grape plants (for example, Vitis spp., Vitis spp. hybrids, and all
members of the subgenera Euvitis and Muscadinia), including scion
or rootstock cultivars. Exemplary scion cultivars include, without
limitation, those which are referred to as table or raisin grapes
Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black
Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose,
Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner,
Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond,
Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic,
Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay,
Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July
Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette,
Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat
Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche,
Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus,
Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson
seedless, and Thomuscat. They also include those used in wine
production, such as Aleatico, Alicante Bouschet, Aligote,
Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc,
Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay (e.g.,
CH 01, CH 02, CH Dijon), Chasselas dore, Chenin blanc, Clairette
blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao
Pires, Flora, French Colombard, Fresia, Furmint, Gamay,
Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green
Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de
Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier,
Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc,
Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino,
Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit
Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George,
Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty,
Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador,
Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert,
Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant,
Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta
Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano,
Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling,
and Zinfandel. Rootstock cultivars include Couderc 1202, Couderc
1613, Couderc 1616, Couderc 3309 (Vitis riparia X rupestris), Dog
Ridge, Foex 33 EM, Freedom, Ganzin 1 (A.times.R #1), Harmony, Kober
5BB, LN33, Millardet & de Grasset 41B (Vitis vinifera X
berlandieri), Millardet & de Grasset 420A, Millardet & de
Grasset 101-14 (Vitis riparia X rupestris), Oppenheim 4 (SO.sub.4),
Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110,
Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A,
Vitis rupestris Constantia, Vitis california, and Vitis girdiana,
Vitis rotundifolia, Vitis rotundifolia Carlos, Teleki 5C (Vitis
berlandieri X riparia), 5BB Teleki (selection Kober, Vitis
berlandieri X riparia), SO.sub.4 (Vitis berlandieri X rupestris),
and 039-16 (Vitis vinifera X Muscadinia).
Using plant tissue culture methods described herein, we have also
developed in vitro selection methods which enable those skilled in
the art to develop pathogen-resistant grapevines. One such
application is the selection of mutations in grape cell cultures.
In this application, cells that are resistant or susceptible to a
particular condition are selected based on increased or selective
growth. The cells can further be exposed to a mutagen that results
in changes in the DNA of the exposed cells. The mutagenized DNA can
then be identified using standard techniques.
A second, related application is the selection of
pathogen-resistant cells. Cells are cultured in the presence of a
phytotoxin from a plant pathogen. Cells that show resistance can
then be used to regenerate a pathogen-resistant plant.
A third application is the transfer of genetic information into
grape cells. The genetic information can include nucleic acid
sequence encoding a selectable marker. Culturing cells in the
presence of the selective pressure (e.g., in the presence of
filtrate from a culture of E. ampelina at a concentration that
kills cells not expressing a nucleic acid of the invention, such as
SEQ ID NO: 6, but does not kill cells that are expressing the
nucleic acid) results in the proliferation or survival only of the
cells that have the desired genetic information. Those in the art
will recognize that determination of the concentration of filtrate
or related compounds may be determined by performing a standard
dose-response assay.
There now follows a description for each of the aforementioned
methods. These examples are provided for the purpose of
illustrating the invention, and should not be construed as
limiting.
EXAMPLE 1
Perennial Grape Embryonic Culture System
The following method has proven effective for the production of
perennial embryogenic grape cultures, and for the regeneration of
grapevine by somatic embryogenesis.
Explant Tissue and Culture Initiation
In the culture initiation step, explant material was collected from
the field, greenhouse, or in vitro shoot micropropagation cultures
of grapevine and placed into in vitro culture. This explant
material was typically collected from leaves, anthers, or tendrils,
but is also obtained from other vegetative or reproductive tissues
of grapevine. Once collected, the explant tissue, if desired, was
surfaced sterilized according to standard methods, and then placed
on a suitable solid culture initiation medium in a petri plate.
Any of a number of well known media, e.g., Murashige and Skoog (MS)
and Nitsch's medium, may be used. Such media typically include
inorganic salts, vitamins, micronutrients, a nitrogen source, and a
carbon source such as sucrose, maltose, glucose, glycerol,
inositol, and the like. For example, sucrose may be added at a
concentration of between about 1 g/L to about 200 g/L; and
preferably at a concentration of between about 30 g/L to about 90
g/L. Moreover, the composition of such plant tissue culture media
may be modified to optimize the growth of the particular plant cell
employed. For example, the culture initiation medium may be
prepared from any of the basal media found Table 1.
TABLE-US-00001 TABLE 1 COMPOSITION OF MEDIA COMMONLY USED IN THE
EXAMPLES Component (mg/ L unless other- wise specified) MS Modified
MS Nitsch KNO.sub.3 1900.0 3033.3 950.0 NH.sub.4NO.sub.3 1650.0 --
720.0 NH.sub.4Cl -- 363.7 -- MgSO.sub.4 7H.sub.2O 370.0 370.0 185.0
CaCl.sub.2 440.0 440.0 166.0 KH.sub.2PO.sub.4 170.0 170.0 68.0
Na.sub.2EDTA 37.23 37.23 37.3 FeSO.sub.4 7H.sub.2O 27.95 27.95
27.95 MnSO.sub.4 H.sub.2O 16.9 16.9 18.9 Kl 0.83 0.83
H.sub.3BO.sub.3 6.2 6.2 10.0 ZnSO.sub.4 7H.sub.2O 8.6 8.6 10.0
Na.sub.2MoO.sub.4 2H.sub.2O 0.25 0.25 0.25 CuSO.sub.4 5H.sub.2O
0.025 0.025 0.025 CoCl.sub.2 6H.sub.2O 0.025 0.025 0.025 Glycine
2.0 2.0 -- Nicotinic acid 0.5 0.5 1.0 Pyridoxin HCl 0.5 0.5 1.0
Thiamine HCl 0.1 0.1 1.0 Inositol 0.1 g/L 1.0 g/L 0.1 g/L Sucrose
30.0 g/L; 60.0 g/L 30.0 g/L; 60.0 g/L; 20.0 g/L 90.0 g/L Activated
Char- -- 0.5 g/L; 1.0 g/L, -- coal 2.0 g/L Agar 7.0 g/L 7.0 g/L 8.0
g/L pH 5.5 5.5 5.5
If desired, the initiation medium may contain an auxin or a mixture
of auxins at a concentration of about 0.01 mg/L to about 100 mg/L,
depending on the cultivar of interest, which is effective for
inducing the production of embryogenic cells or embryogenic cell
masses on the explant tissue. For example, explant tissue can be
maintained on an agar-solidified Nitsch's-type medium supplemented
with, for example, between about 0.01 mg/L and about 10 mg/L of
2,4-D, and preferably between about 0.5 mg/L and about 3.0 mg/L of
2,4-D. 2,4-D is just one example of an auxin which is useful in the
methods of the invention. Other auxins include, for example, NAA,
NOA, IAA, dicamba, and picloram. Additionally, if desired, other
plant growth regulators may be included in the medium at standard
concentrations. For example, cytokinins (e.g., a
naturally-occurring or synthetic cytokinin, such as BA or zeatin),
if present, may be used at a concentration of from about 0.01 mg/L
to about 10 mg/L, and preferably about 0.3 mg/L, depending on the
cultivar of interest. In some instances, other classes of growth
regulators, such as ABA or GA, may be included at appropriate
standard concentrations. For example, ABA may be added at a
concentration of about 0.5 mg/L to about 20 mg/L, and preferably at
a concentration of about 5 mg/L; and GA may be added at a
concentration of about 0.1 mg/L to about 30 mg/L, and preferably at
a concentration of about 5 mg/L. The addition of plant growth
regulators at this stage is not necessary for the induction of
embryogenesis. Additionally, the initiation medium may also include
activated charcoal (0.1 2.0 g/L) or a similar adsorbent known to
those in the art.
Culturing of explant tissue during this stage is preferably carried
out in the dark at 22 30.degree. C., although it may also be
carried out under very low light conditions, or in full light.
After approximately one to four weeks in culture, explant tissue
cultures are then placed in full light with a 16 hr photoperiod.
Cultures are scanned weekly for the presence of emerging
embryogenic cells or embryogenic cell masses. Embryogenic cells or
cell masses are identified based on morphology. Embryogenic cell
masses, in general, tend to be white to pale yellow in color, and
are often hyaline. They may be recognized from a very early, small
stage (10 20 cell aggregates), based upon their color and friable,
granular appearance. Embryogenic cultures are also identified by
their compact nature with cells that are rich in cytoplasm (as seen
under the microscope). The embryogenic cultures appear at varying
frequencies depending on a multitude of factors including, but not
limited to, genotype, nature and type of explant, medium
composition, and season of harvest. Careful visual selection to
ensure transfer of appropriate embryo-like structures is required
for culture maintenance. Once identified, embryogenic cells or cell
masses are then transferred to culture maintenance medium, as
described herein.
Culture Maintenance
Embryogenic cells or embryogenic cell masses are carefully removed
and transferred to a culture maintenance medium. Again, any of a
number of well known media, e.g., MS and Nitsch's medium, may be
used. Although not generally required, plant growth regulators may
be added as described above.
In general, embryogenic tissue can be maintained by subculturing at
regular intervals (e.g., every one to four weeks, or every four to
eight weeks) to new maintenance medium, as described herein.
Alternatively, embryogenic tissue can be placed in a liquid culture
medium (e.g., MS, B-5, or Nitsch) and grown as a liquid embryogenic
suspension as described herein. Embryogenic cell masses are grown
to increase embryogenic cell biomass as required by division of
expanding cultures during transfer. The cultures can be prompted to
develop toward increasing embryogenesis or toward less
embryogenesis and more unorganized embryogenic cell growth by
repeated manipulation of the culture, which includes careful
selection of embryogenic cells and cell masses during transfer.
Repeated transfer of embyogenic cells or cell masses has not only
been found to enrich the growth of embryogenic tissue, but also to
facilitate the process of somatic embryogensis. The cultures are
perennial in that they typically persist for over two years.
A key component of the present approach involves the careful
selection of embryogenic cells from explanted tissue, followed by
recurrent selection and subculturing of the selected embryogenic
tissue. This material has not only been found to be useful in the
regeneration of whole grape plants from somatic embryos, but has
also been found to have a significantly increased capacity for
embryogenesis, including the production of somatic embryos. By
carrying out this procedure, the growth of embryogenic cells is
enriched, speeding the process of somatic embryo formation and
subsequent plant regeneration.
The explant material taken from plants that were grown from somatic
embryos was observed to exhibit an enhanced embryogenic potential,
when compared to explant material taken from clonal explant tissue
which had not been cultured for the production of embryogenic
cells. This increase in embryogenic potential was observed to
increase after two or more successive initiation, culture and plant
regeneration cycles (e.g., clonal
plant-->explant-->embryogenic culture initiation-->somatic
embryo-->somatic embryo-derived plant-->explant). It is not
necessary to use a somatic embryo-derived plant as the source of
the explant; somatic embryos or even embryogenic cultures that have
been transferred to new medium will also produce new somatic
embryos with increased embryogenic potential. Such explant material
is conveniently maintained as in vitro axillary shoot cultures,
which serves as the source for vegetative explants; however, other
methods of plant maintenance are also acceptable.
Germination and Plantlet Growth
Somatic embryos obtained from the above-described cultures are
subsequently germinated into grape plantlets according to standard
methods. For example, somatic embryos are placed on the surface of
a germination medium (e.g., MS medium) in sterile petri plates. The
cultures containing the embryos are incubated in a growth chamber
under lighted conditions (16 hr photoperiod). During germination
the root emerges and the epicotyl begins to grow. When grape
plantlets that are grown on germination medium reach sufficient
size (1 cm, with at least two leaves), they can be removed from the
culture dishes and planted in a sterilized potting mixture.
Plantlets are typically transferred into nursery containers in a
soiless potting mix (e.g., Vermiculite, Perlite, or ProMix.TM., V.
J. Growers, Apopka, Fla.). If desired, plantlets can be placed in a
growth chamber or in a greenhouse moisture chamber and incubated
under high humidity conditions (90% humidity) for plantlet growth
and acclimatization. Subsequently, acclimatized plantlets can be
transferred outdoors to a vineyard or to a greenhouse.
In one example, we describe the production of an embryogenic
perennial culture of Vitis vinifera cv. `Thompson Seedless.` Mother
plant-derived cultures were obtained from a leaf that was
surface-disinfected and inoculated onto culture initiation medium
described by Nitsch (1968) and modified by Gray D. J. ("Somatic
Embryogenesis in Grape." In: Somatic embryogenesis in woody
perennials, Vol. 2, Gupta P. K., Jain S. M., and Newton R. J.
(Eds.), Kluwer Academic, Dordrecht, The Netherlands, pp. 191 217,
1995). This medium contained about 1.1 mg/L of 2,4-D and about 0.05
mg/L of BA. After explanting the tissue, the culture vessels were
incubated in complete darkness for six weeks. Most of the explanted
tissue was observed to form a mass of undifferentiated, highly
vacuolated cells within this six week period. Embryogenic cultures,
identified by their compact nature and the presence of cells that
were rich in cytoplasm, were repeatedly subcultured. The resulting
culture was obtained by the selection method described above,
followed by subculturing (for about 6 weeks) until enough
embryogenic culture was available. A somatic embryo originally
obtained from the mother plant (i.e., 1.sup.st generation embryo)
was germinated, and its shoot tip used to create an in vitro
micropropagation culture. Leaves from the plant derived from that
culture were then used to produce a new embryogenic culture
(2.sup.nd generation). A somatic embryo obtained from that culture
was similarly used to create the third generation. The embryogenic
response of Vitis vinifera cv. `Thompson Seedless` from in vitro
micropropagation culture-derived leaves is presented in Table 2.
These results show the comparison of leaves from potted mother
plant-derived cultures with leaves from plants derived from
cultures obtained from third-generation germinated somatic
embryos.
TABLE-US-00002 TABLE 2 No. leaves No. embryogenic % response
Culture derivation cultured cultures per leaf Mother plant 195 0* 0
3.sup.rd generation somatic 200 14 .sup. 7.sup.# embryo *Other
experiments have yielded one embryogenic culture. .sup.#The percent
response per leaf has been found to be has high as 30%.
In addition, perennial embryogenic cultures from other grapevines
have also been produced using the methods described herein,
including Vitis longii, Vitis rotundifolia (cv. Carlo and Dixie),
Vitis rupestris, Vitis vinifera (cv. Autumn Seedless, Cabernet
Sauvignon, Cabernet Franc, Chardonnay, Dolcetto, Gamay Beaujolais,
Lambrusco, Pinot Noir, Semillon, Tokay, White Riesling, Zinfindel,
and the like), and several Vitis hybrids (cv. Blanc du Bois,
Niagara Seedless, Seyval Blanc, Stover, Southern Home and the
like).
EXAMPLE 2
Production of Highly Embryogenic Grape Cells Using Liquid
Suspension or Solid Cultures
A method has also been developed for the production of large
quantities of grapevine somatic embryos using either a liquid cell
suspension culture or a solid culture system. These methods are
particulary useful for producing highly embryogenic cells that are
capable of regenerating into whole plants. Below, a simple protocol
for efficient somatic embryogenesis of grapevine using either a
liquid cell suspension culture or a solid culture system is
presented.
In general, the method includes a multistage culturing process
typically involving (i) culture initiation; (ii) identification and
isolation of embryogenic cells or embryogenic cell masses; (iii)
production of perennial embryogenic cultures; and (iv)
concentration of highly embryogenic cell clusters. The method
involves the following steps.
Explant tissue is placed on a suitable culture initiation medium,
as is described herein. After approximately six weeks on culture
initiation medium, embryogenic cells and embryogenic cell masses
are identified. Once identified, embryogenic cultures, which may be
less than 1 mm in diameter, are isolated and cultured on fresh
initiation medium to encourage growth, as described herein.
Subculturing of the embryogenic cultures typically results in the
formation of somatic embryos. Embryogenic cultures and early stage
somatic embryos obtained from these cultures are then further
cultured in a suitable liquid plant growth medium. For example, the
plant tissue culture nutrient media, consisting B-5 medium (Gamborg
et al., Exp. Cell. Res. 50:151 158, 1968; Sigma Chemicals, St.
Louis, Mo.), that has been modified as described by DeWald et al.
(J. Amer. Soc. Hort. Sci. 114:712 716, 1989) and Litz et al.
("Somatic embryogenesis in mango," 1995, supra). This modified
medium consists of B-5 major salts, MS minor salts and vitamins,
glutamine (about 400 mg/L), and sucrose or commercial table sugar
(about 60 g/L). Before autoclaving, the pH of the medium is
adjusted to about 5.8. Although 2,4-D (about 0.5 2.0 mg/L) is the
preferred growth regulator used in this medium, other growth
regulators, such as, for example, dicamba, picloram, NOA, or
2,4,5-trichlorophenoxy acetic acid, may be also used at appropriate
concentrations, for example, those described above. Flasks
containing the embryogenic cell cultures, somatic embryos, or both
are subsequently incubated at about 26.degree. C. on a rotary
shaker at 125 rpm in darkness or diffuse light. The cultures are
then subcultured as described herein, typically once every ten to
fourteen days, but subculturing regimens may vary depending on the
growth and proliferation of embryogenic cell clusters.
In about six to eight weeks, a fine cell suspension culture is
produced, which consists of highly-vacuolated elongated cells
(non-embryogenic cells), and also a lesser number of small,
cytoplasm-rich, isodiametric cells (embryogenic cells). Once
sufficient culture is produced, the differentiated embryos can be
removed from the culture by sieving, and the differentiated embryos
are discarded. Continued maintenance of the sieved embryogenic cell
suspension culture in modified B-5 liquid medium, with periodic
subcultures, has been found to increase the biomass of embryogenic
cell clusters.
After approximately twelve to sixteen weeks, a large mass of highly
concentrated embryogenic grape cells is typically observed. The
time taken for the concentration of embryogenic cells or
embryogenic cell masses may vary depending on several factors,
including the cultivar, genotype, and culture conditions.
Embryogenic cells at this stage are especially useful in virtually
any type of genetic transformation method. These embryogenic cells
can also be induced to differentiate into somatic embryos according
to any standard method, e.g., by culturing the cells in modified
B-5 liquid medium devoid of growth regulators for a period of about
four to six weeks. Alternatively, the early stage somatic embryos
may be plated in medium solidified by the addition of a suitable
gelling agent such as gellan gum, agarose, agar, or any other
similar agent, for further differentiation of somatic embryos in
complete darkness. If desired, torpedo/cotyledonary-stage embryos
can be individually subcultured on a standard maturation medium,
e.g., a maturation medium consisting of MS nutrient formulations.
Mature somatic embryos are then transferred to a growth chamber for
germination, and regeneration to plants in an appropriate
container. The frequency of somatic embryo formation using this
procedure is typically high.
There now follows a description of the results for the production
of embryogenic cells and cell masses obtained from a liquid
suspension and solid cultures of `Thompson Seedless` and two
different clones of `Chardonnay,` CH 01 and CH 02.
Asynchronous somatic embryos of Vitis vinifera cv. `Thompson
Seedless` and `Chardonnay` CH 01 and CH 02 obtained from perennial
embryogenic tissue were further cultured in a liquid medium to
produce a callus tissue suspension culture. After about fourteen
days and following about two to three subcultures (a subculture was
performed about every fourteen days), an amorphous, yellowish to
creamy white colored callus was produced. As a result of the
production of callus tissue, the liquid culture media in the tissue
culture flask appeared as a dense suspension. Microscopic
examination revealed that the callus cells were elongated and
highly vacuolated, and exhibited no signs of embryogenic capacity.
Amorphous callus continued to proliferate, even when the somatic
embryos used to initiate the culture were removed from the
culture.
After approximately six weeks in modified B-5 liquid medium, we
observed the production of small clusters of cytoplasm-rich cells
as white clumps (FIG. 1A). These embryogenic masses were observed
to proliferate exponentially, and grew to the capacity of the flask
in about ten to twelve weeks.
Continued maintenance of these embryogenic masses as a single unit
(i.e., in one flask) is often detrimental, as the cultures have
been found to deteriorate in quality, and eventually turn brown.
Dividing these embryogenic cultures into smaller units during
subculturing assists to proliferate and increase the biomass of the
divided cultures. Among the two cultivars tested, both clones of
`Chardonnay` were found to be equally fast growing and outgrew
`Thompson Seedless.` While the embryogenic masses of `Chardonnay`
were creamy white to yellowish in color, those of `Thompson
Seedless` were dull white or brownish. In addition, `Thompson
Seedless` appeared to be more sensitive to culture density, as the
cells were observed to turn dark if the culture density was not
corrected. The preferred culture density was approximately 400 mg
of embryogenic cells per 40 mL of liquid modified B5 medium in a
125 mL flask.
Somatic Embryo Production in Liquid Culture
Embryogenic masses were passed through a 960 micron nylon sieve and
collected in a sterile beaker. Sieving of the embryogenic masses to
initiate embryogenesis in liquid culture was found to serve two
purposes. First, a fair degree of synchronization of embryo
differentiation was obtained. Second, the formation of somatic
embryo abnormalities during differentiation, such as fasciation or
fusion, was reduced. After four to six weeks in liquid medium,
small, white somatic embryos in the globular or early heart stage
were observed. Sieving the cultures at this stage did not
facilitate an increase in embryo differentiation.
After approximately eight weeks, somatic embryos were clearly
visible, and a few embryos were found to have reached the
cotyledonary stage of embryo development. Sieving the
differentiated embryos and culturing them in a separate flask,
however, facilitated faster differentiation, as well as
synchronization of embryo devlopment.
Both `Chardonnay` clones--CH 01 and CH 02--were found to readily
differentiate into somatic embryos. Appropriate sieving and density
adjustment (performed by culturing about 1000 mg of somatic embryos
per 40 mL medium) ensured greater synchronization and singulation,
as well as embryo differentiation (FIG. 1B). In approximately
twelve to fourteen weeks after subculture in liquid embryogenesis
medium, singulated somatic embryos started to turn green and
radicles elongated, showing the onset of precocious germination
(FIG. 1C).
Cultures of `Thompson Seedless` initially were found not to advance
beyond the heart stage in liquid culture. In addition, the embryos
were found to be more clustered, often resulting in the formation
of fused somatic embryos. Removal of the abnormal embryos and
lowering the culture density by half resulted in normal somatic
embryogenesis in liquid culture. These somatic embryos reached
maturity in about fourteen to eighteen weeks.
Somatic Embryo Production in Solid Medium
Embryogenic cells or embryogenic cell masses obtained from liquid
cultures were observed to differentiate into somatic embryos as
early as three weeks after culture initiation. After four weeks of
culture, microscopic examination also revealed the formation of
globular and heart shaped somatic embryos on the callus tissue
(FIG. 1D). The somatic embryos were hyaline, and resembled that of
a hyperhydric state (FIG. 1E); however, the embryos continued to
differentiate, and were found to develop into mature somatic
embryos in another three to four weeks. These somatic embryos were
observed to develop a suspensor (FIG. 1E). In addition, embryogenic
cells were observed to develop into a mass of asynchronous somatic
embryos.
One of the interesting observations from these experiments was that
the majority of somatic embryos arose as individual units, and not
as small clumps, although there were a few clumps of somatic
embryos. In such cases, the number of somatic embryos ranged from
six to ten in each clump, and these embryos were easily separated
from the callus tissue. Embryos found in the cotyledonary stage
were isolated on a weekly basis, and subcultured for maturation.
Each clump of embryogenic mass continued producing somatic embryos
for at least twelve weeks. Embryogenic cell masses tended to turn
brown in solid medium, containing Gel-Gro (ICN Biochemicals), but
this discoloration did not adversely affect culture viability.
About four or five weeks later, clusters of somatic embryos started
to appear on the surface of the brown embryogenic cells or
embryogenic cell masses.
Somatic Embryo Maturation, Germination and Plant Regeneration
Three maturation media--mango maturation medium (Litz et al., 1995,
supra); mango maturation medium solidified with agar (7 g/L); and
MS basal medium with 3% sucrose--were studied to evaluate the
ability to promote somatic embryo germination and plant
regeneration. Our results indicated that a MS basal medium
containing 3% sucrose was the most effective at promoting embryo
maturation, germination, and plant regeneration, for both embryos
derived from solid medium and for the precociously germinated
embryos that were obtained from liquid medium cultures (FIG. 1D).
Although there was good germination on mango maturation medium with
agar, the quality of the regenerants was not as good as with MS
salts with 3% sucrose. Embryos from the two systems studied (i.e.,
liquid and solid media) showed variation between themselves in
germination and regeneration. Although embryos have precociously
germinated in liquid cultures, continued germination in these
cultures was not observed. On transfer to solid medium, however,
the embryos were found to continue the germination process, and
resulted in the formation of grape plants with a dense root system.
Continued maintenance in liquid medium after radicle emergence lead
to hyperhydricity and eventually plant regeneration was reduced
from these embryos. Accordingly, it is preferred that the somatic
embryos should be removed from the liquid as soon as they
precociously germinate and transferred to solid medium.
Long-Term Preservation of Suspension-Derived Grapevine Somatic
Embryos and Regeneration of Plants
We have established a method for the long-term storage of somatic
embryos. Mature somatic embryos from suspension cultures of
`Chardonnay` were blot-dried on sterile filter paper in a
laminar-flow hood and then stored in sterile petri plates at
6.degree. C. Samples were periodically drawn from these plates and
germinated on MS medium with 3% sucrose. Germination (i.e., the
emergence of roots from the somatic embryo) and plant regeneration
were recorded. Table 3 shows the data from clone CH 02 after 22
months in storage, and Table 4 shows the data from clone CH 01
after 5 months in storage.
TABLE-US-00003 TABLE 3 Trial Number of Number Germinated Number of
Percent Number Embryos (Percent Germinated) Plants Yield 1 87 81
(93.1) 69 79.3 2 42 40 (95.2) 35 83.3 3 41 41 (100.0) 30 73.2 Total
170 162 (95.3) 134 78.8
TABLE-US-00004 TABLE 4 Trial Number of Number Germinated Number of
Percent Number Embryos (Percent Germinated) Plants Yield 1 15 15
(100.0) 13 86.7 2 15 15 (100.0) 13 86.7 3 15 13 (86.7) 9 60.0 4 15
12 (80.0) 7 46.7 5 15 13 (86.7) 9 60.0 6 15 11 (73.3) 9 60.0 7 15
15 (100.0) 14 93.3 8 15 13 (86.7) 9 60.0 Total 120 107 (89.2) 83
69.2
Direct Seeding of Suspension Culture-Derived Grapevine Somatic
Embryos
`Chardonnay` and `Thompson Seedless` grapevine somatic embryos were
produced from liquid cultures as described herein.
Suspension-derived, mature somatic embryos were blot dried briefly
in the laminar flow hood and germinated directly in Magenta vessels
containing one of the following potting media: i) sand; ii)
ProMix.TM. commercial potting mixture (CPM); or CPM overlaid with
sand. Each vessel containing 20 mL of distilled water and the
potting medium was sterilized by autoclaving for 30 min and cooled
overnight prior to inoculating the somatic embryos. Three somatic
embryos were placed in each vessel. Seeding was carried out under
aseptic conditions and the containers were closed and incubated at
26.degree. C. with a 16 hr photoperiod at 75 .mu.mol s.sup.-1
m.sup.-2 light intensity. Results revealed that CPM overlaid with
sand was ideal for plant development. Although sand promoted more
germination, the resulting plants were chlorotic and their survival
rate was poor. There was more contamination of somatic embryos on
pure CPM. The present study offers scope for large-scale
multiplication of grapes using suspension cultures and sets the
platform for growing grape somatic embryos in bioreactors.
The experimental results described above were carried out using the
following techniques.
Culture Initiation
Embryogenic cultures were initiated from anthers and ovaries of the
cultivar "Chardonnay" (Clones CH 01 and CH 02), and from the leaves
of the cultivar "Thompson Seedless" according to standard methods,
e.g., those described herein. Somatic embryos of these cultures,
initiated and maintained in modified MS medium, were used to
initiate liquid cell suspension cultures. Typically these cultures
are highly asynchronous in embryonic development and
differentiation and, therefore, each inoculum consisted of somatic
embryos at various stages of development.
Establishment of Liquid Cultures from Differentiated Somatic
Embryos
The composition of the liquid medium was adapted from the medium
described by Litz et al., supra as follows. Callus induction was
achieved by the addition of 1 mg/L of 2,4-D in the medium. The pH
of the medium was adjusted to about 5.8, and dispensed as 40 mL
aliquots in 125 mL Erlenmeyer flasks. The flasks were tightly
covered with heavy duty aluminum foil before autoclaving. After
cooling, approximately one gram of the somatic embryos was
transferred to the liquid medium using a sterilized spatula under
aseptic conditions. The neck of the flask was sealed with Parafilm,
and the cultures were then incubated in semi-darkness (diffused
light) on a rotary shaker at about 120 rpm. The cultures were
subcultured at least one time every two weeks.
Flasks containing the suspension cultures were removed from the
orbital shaker and the cultures were allowed to settle for about 15
minutes. The supernatant was gently decanted into a sterile flask,
leaving the embryogenic cells in a minimal volume (approximately 5
mL). Approximately 35 mL of fresh liquid medium was added to the
embryogenic cells and swirled quickly. The entire contents of the
flask were then transferred to a sterile 125 mL flask. This second
flask, containing the embryogenic cells, was then sealed with
Parafilm and returned to the orbital shaker.
The amorphous callus generated from the somatic embryos was
collected as follows. The embryogenic suspension, including
differentiated somatic embryos and callus, was allowed to settle in
the flasks. About half of the supernatant medium was decanted, and
the remainder was swirled and quickly filtered through a
presterilized, nylon mesh (960 microns), placed over a 150 mL
beaker. While the differentiated somatic embryos were retained in
the mesh, the fine callus that passed through along with the liquid
medium was collected in the beaker. The callus that was collected
in the beaker was next filtered through a sterile, double-folded,
Kimwipe placed over a sterile funnel. The amorphous callus that
adhered to the Kimwipe was subsequently removed from the Kimwipe
using a sterilized spatula, and resuspended in fresh liquid culture
medium. Approximately 100 mg of the callus was suspended in each
flask. These liquid cultures were subcultured as described herein
approximately once every fourteen days in modified B-5 liquid
medium containing 2,4-D.
Somatic Embryo Production in Suspension Culture
Embryogenic cells or cell masses that were initiated in liquid
suspension cultures were sieved using a 960 micron sieve, and the
finer fraction was harvested in liquid embryogenesis medium, under
aseptic conditions. The medium composition was the same as that of
the initiation medium; however, 2,4-D was omitted from the medium
and about 0.05 mg/L of BA was added. After adjusting the pH to 5.8,
the medium was dispensed as 40 mL aliquots in 125 mL Erlenmeyer
flasks, covered with aluminum foil and autoclaved. Approximately
100 mg of callus was cultured in each flask. The cultures were
maintained in semidarkness at 25.degree. C. on a rotary shaker at
120 rpm, and subcultured once every 14 days. Sieving of cultures
was done as necessary, in order to synchronize differentiated
somatic embryos. Finer mesh sieves (e.g., 520 micron sieves), if
necessary, may also be employed.
Germination of Somatic Embryos from Suspension Cultures and
Regeneration
Greening somatic embryos having elongated radicles were sieved from
the suspension cultures. Somatic embryos were individually picked
and cultured. Three different media--mango maturation medium (Litz
et al., supra), mango maturation medium solidified with agar (7
g/L) instead of Gel-Gro, and MS basal medium with 3% sucrose--were
tested for germination and plant regeneration. Plant growth
regulators were omitted from these media preparations. Twenty-five
embryos were cultured in each standard petri plate, and eight
plates of each medium was tested. After sealing with Parafilm, the
cultures were incubated in a growth chamber under a 16 hour
photoperiod. Plantlets with four true leaves were subsequently
transferred to soil.
Somatic Embryo Production in Solid Medium
Embryogenic cells and embryogenic cell masses produced in
suspension cultures were harvested as described above and then
transferred to solid embryogenesis medium. The medium consisted of
the same compounds as the liquid embryogenesis medium, and
solidified with 2.0 g/L Gel-Gro or 7 g/L agar. Approximately 50 mg
of callus was placed as a clump onto a medium-containing petri
plate and each plate had two such clumps. After inoculating, the
petri plates were sealed with Parafilm and incubated in complete
darkness. Subculturing was performed after somatic embryo
differentiation was observed. Somatic embryos produced from the
embryogenic cells or embryogenic cell masses were counted on a
weekly basis, starting from six weeks after culture. Embryos of
cotyledonary stage were counted and subcultured for maturation.
Maturation and Germination of Somatic Embryos from Solid Medium
Mature somatic embryos that were approximately 5 mm in length were
isolated from the asynchronous mass and cultured on maturation
medium. Twenty-five mature somatic embryos were cultured in each
standard petri plate on MS medium with 3% sucrose. The cultures
were kept in the dark until they germinated. After elongation of
radicle, they were transferred to light under a 16 hour
photoperiod. Plantlets with at least four true leaves were
subsequently transferred to soil.
EXAMPLE 3
Selection of Disease Resistant Embryogenic Cells and Plants of
Grapevine
The perennial grape embryogenic cultures of the invention can be
used for the selection or screening for grape cells having
resistance to toxic substances, such as those present in a filtrate
produced by a fungal culture. Such pathogens include, without
limitation, bacteria and fungi. Plant diseases generally caused by
these pathogens are described in Chapters 11 16 of Agrios, Plant
Pathology, 3rd ed., Academic Press, Inc., New York, 1988, hereby
incorporated by reference. The "Compendium of Grape Diseases" (APS
Press (1988) R. C. Pearson & A. C. Goheen, Eds.) describes
diseases that affect grape plants. Examples of bacterial pathogens
include, without limitation, Agrobacterium vitis, Agrobacterium
tumefaciens, Xylella fastidosa, and Xanthomonas ampelina. Examples
of fungal pathogens include, without limitation, Plasmopara
viticola, Botrytis cinerea, Guignardia bidwellii, Phomophsis
viticola, Elsinoe ampelina, Eutypa lata, Armillaria mellea, and
Verticllium dahliae. Others are described herein.
By exposing embryogenic cultures to a phytotoxin (e.g., crude
culture filtrate or a purified phytotoxin obtained from a plant
pathogen), resistant grape cells can be selected and propagated.
Grape cells that survive the selection pressure are expected to
resist not only the selecting toxin, but also the original microbe
that produces the toxin. Moreover, due to the dynamics of the
selection process, induced resistance may also function against an
array of disease-causing organisms beyond the original microbe used
for selection. Because the selection is carried out at the cellular
level, it is likely that grape plants regenerated from the cells
will show the selected characteristic. In particular, this system
allows one skilled in the art to select or screen for the desired
characteristic from among thousands of cells in a single culture
flask or petri plate.
EXAMPLE 4
Methods for Selecting Pathogen-Resistant Somatic Embryos and
Producing Plants
Various microbes attack grapevine and cause a number of diseases.
These diseases include fungal diseases of leaves and fruits (such
as black rot and anthracnose), fungal diseases of the vascular
system and roots (such as Esca, Black Measles, Black Dead Arm, and
Eutypa dieback) and bacterial diseases (such as crown gall and
Pierce's disease).
One disease affecting grapevine is anthracnose, also known as
bird's eye spot disease, which is caused by the fungus, E.
ampelina. Under favorable conditions, this fungus attacks almost
all the aerial parts of the grapevine, including fruits, causing
extensive damage to the crop. Anthracnose causes the appearance of
circular lesions with brown or black margins and round or angular
edges on the grapevine plant. The center of the lesions becomes
grayish white and eventually dries up and falls off, leaving a
`shot-hole` appearance. The disease especially affects young
leaves, preventing normal development. New shoots are also affected
and acquire an obvious, burnt appearance. Fruit clusters are also
susceptible to fungal infection throughout their development;
lesions on the berries extend into the pulp, often inducing
cracking.
Preparation of Phytotoxin
An E. ampelina culture filtrate having toxic activity was prepared
as follows. Full-strength Czapekk-Dox broth medium (Fisher
Scientific, Springfield, N.J.) was prepared by dissolving the
required amount of the broth mixture in deionized (DI) water. The
medium was dispensed as 50 mL aliquots in 125 mL Erlenmeyer flasks.
After autoclaving and cooling, 100 .mu.L of a E. ampelina spore
suspension was added to each flask (Day 1); the flask was then
incubated in a rotary shaker at 25.degree. C. at 120 rpm for one
week in the dark. After one week, the contents in each flask were
transferred to 100 mL of full strength Czapek-Dox in a 250 mL
Erlenmeyer flask and the incubation was continued for two more
weeks. At the end of this period (i.e., three weeks from Day 1),
the fungal culture filtrate was collected by filtering the contents
of each flask through a sterile, multi-layer cheese cloth. The
crude culture filtrate was stored at -4.degree. C. until further
use.
Prior to addition to the culture media for in vitro selection, the
frozen culture filtrate was thawed (without heating) at room
temperature, pH adjusted to 5.8, and filter-sterilized through a
0.2 micron filter (Nalgene, Rochester, N.Y.). This
filter-sterilized pathogen filtrate was found to retain its toxic
activity, as determined by its ability to cause grape plant cell
death.
Selection
The E. ampelina culture filtrate was next added to liquid
suspension cultures of V. vinifera cv. `Chardonnay` embryogenic
cells and embryogenic cell masses in modified B-5 medium to select
cells having resistance to the toxic fungal culture filtrate. The
grape embryogenic cells and embryogenic cell masses were grown as
described above; however, in this in vitro selection, the medium in
which the cells were grown was supplemented with known volumes of
E. ampelina culture filtrate. Appropriate dilutions of pathogen
filtrate were determined by examining the toxicity of the filtrate
using serial dilution analysis. These experiments demonstrated that
a 40% (v/v) culture filtrate was useful for in vitro selection.
Cultures of `Chardonnay` embryogenic cells and embryogenic cell
masses were maintained in liquid medium containing 40% (v/v) fungal
culture filtrate at about 26.degree. C. on a rotary shaker (125
rpm) in diffuse light. Subculturing was done once every ten days;
during each subculture, filter-sterilized culture filtrate was used
to dilute the medium. Selection with culture filtrate was continued
for four or five cycles (each cycle=ten days) of subculture. While
most of the embryogenic cells died, a very few cells, often less
than 1%, survived the selection pressure. Resistant culture lines
were established by withdrawing the selection pressure after four
or five cycles and letting the surviving cells grow in modified B-5
medium devoid of culture filtrate. These resistant lines were
proliferated, and somatic embryo were produced using the methods
described herein.
Embryogenic cell cultures obtained from the selection process were
subsequently tested for resistance to E. ampelina using a number of
in vitro bioassays. We first analyzed whether the resistant
grapevine lines were producing an activity that could inhibit the
growth of the fungus. To this end, the culture medium (i.e.,
conditioned culture medium) from a resistant culture line was
tested for an inhibitory activity against the fungus. Conditioned
media was collected from different cell cultures having resistance
to E. ampelina, and used in several concentrations to prepare
fungal growth media. An actively growing mycelial colony was placed
in the center of a petri plate containing a fungal growth medium
prepared with or without (control) conditioned medium from a
resistant grapevine culture line and incubated under standard
conditions. The results of these experiments showed that the growth
of the fungus was inhibited by a fungal growth medium containing
25% or more of the conditioned medium.
To further demonstrate the presence of anti-fungal activity in
resistant grapevine cultures, resistant lines, as well as control
lines, were placed in solid plant growth medium in six and twelve
o'clock positions in petri plates, and incubated in darkness for
four weeks. After this period, a plug of mycelium from an actively
growing E. ampelina colony was placed in the center of the petri
plates and incubated under standard conditions. The fungus was
observed to grow rapidly and infect the control cultures.
Conversely, fungal growth was inhibited on the plates containing
grape cell cultures having resistance to the E. ampelina culture
filtrate. Hyphae did not grow freely through the medium in plates
containing these resistant cultures, as compared to fungal hyphae
growth through the medium in plates containing control (i.e.,
non-resistant) cultures. A thick mat of mycelium, as seen in the
plates containing control cultures, was never formed in the plates
containing the E. ampelina resistant cultures. This capacity of the
resistant cultures to inhibit the growth of E. ampelina was
retained nine months after selection, demonstrating that the
genetic changes in the resistant cultures were stable.
In addition, the grapevine cultures that were resistant to E.
ampelina were tested for resistance to a second fungal pathogen,
Fusarium (F.) oxysporum. Resistant lines, as well as control lines,
were placed in solid plant growth medium in six and twelve o'clock
positions in petri plates, and incubated in darkness for four
weeks. After this period, a plug of mycelium from an actively
growing F. oxysporum colony was placed in the center of the petri
plates and incubated at room temperature under a 16 hour
photoperiod. The fungus was observed to grow rapidly and infect the
control cultures. Conversely, growth of F. oxysporum was inhibited
on the plates containing grape cell cultures having resistance to
the E. ampelina culture filtrate. Hyphae did not grow freely
through the medium in plates containing these resistant cultures,
as compared to fungal hyphae growth through the medium in plates
containing control (i.e., non-resistant) cultures. A thick mat of
Fusarium mycelia, as seen in the plates containing control
cultures, was never formed in the plates containing the E. ampelina
resistant cultures. This experiment demonstrated that the resistant
grapevine cultures were not only resistant to E. ampelina, but were
also resistant to F. oxysporum.
Further analysis was made to determine if the fungal-resistant
grapevine cultures could give rise to somatic embryos that were
also resistant to E. ampelina. Somatic embryos derived from
resistant cultures and control cultures were grown either in medium
containing 40% (v/v) of fungal culture filtrate or in control
medium containing no fungal culture filtrate. While somatic embryos
derived from the resistant cultures formed and germinated normally
in both the fungal culture filtrate-containing medium and control
medium, somatic embryos derived from control cultures turned
necrotic and eventually died in the fungal culture
filtrate-containing medium, but did not die in the control medium.
The necrosis of the controls in the fungal culture
filtrate-containing medium was rapid enough to turn the control
somatic embryos dark within seventy-two hours of culture
initiation. The results from these experiments demonstrated that
the somatic embryos obtained from resistant cell cultures were also
resistant to the fungal filtrate. Furthermore, these resistant
somatic embryos were observed to withstand a concentration of E.
ampelina culture filtrate that was equal to that withstood by their
progenitor resistant embryogenic cells and embryogenic cell
masses.
Pathogen-Resistant Plants
Embryogenic cultures were selected in vitro against fungal culture
filtrate produced by E. ampelina. Plants were regenerated from the
selected cultures and acclimatized in the greenhouse. Plants from
selected lines and unselected controls were sprayed with a spore
suspension (1.times.10.sup.6 spores/mL) until runoff. The plants
were individually bagged to maintain humidity (a condition is
optimum for the pathogen to cause anthracnose disease) for 3 days.
The bags were then removed and the plants were scored for
anthracnose symptoms. All of the unselected controls exhibited a
very high degree of susceptibility, and in most cases there was
defoliation due to the disease within three days. Among the 40
different plants from the two selected lines, only one plant showed
mild anthracnose symptoms. These data show that the resistance
acquired by the embryogenic cells during in vitro selection can be
translated into whole plant resistance against the pathogen.
In Vitro Selection and Establishment of Resistant Lines
PEMs became brown and necrotic within a few days of culture in
culture filtrate-containing medium. The medium also turned dark
brown in these flasks. As selection progressed, browning of the
medium was gradually reduced, which was accompanied by necrosis of
most of the PEMs. Only a few PEMs (or cells within a few PEMs)
survived selection pressure through four or five cycles of
selection (FIG. 2). Cultures that survived four and five cycles of
selection were designated as `resistant culture 1` (RC1) and
`resistant culture 2` (RC2), respectively. By continuous
subculturing of these resistant cultures in suspension, we
increased the tissue mass in approximately 5 months after
withdrawing selection pressure. These cultures were used in
subsequent studies and for plant regeneration. There was no
browning in cultures that were grown in medium containing 40% (v/v)
of Czapek-Dox broth. PEMs in these flasks grew normally as in the
non-selected controls. This indicates that the necrosis was caused
by compounds, that were produced by the fungus and released into
the culture filtrate.
Dual Culture
Mycelium of E. ampelina grew uninhibited on plates containing PEMs
and somatic embryos from non-selected control. Within a week after
fungal inoculation, mycelium covered the entire plate, growing on
the embryogenic tissue as well. However, both selected lines (RC1
and RC2) inhibited the growth of mycelium significantly (FIG. 3A).
Even after 10 days, the mycelial growth did not reach the PEMs. A
clear zone of inhibition could be observed for several days. A
similar trend was observed with F. oxysporium, which is not a
pathogen of grapevine (FIG. 3B). Mycelial growth was white, fluffy
and rapid on the non-selected controls. On the selected lines,
however, the fluffy growth was restricted to the central region of
the plate. There was more vertical mycelial growth compared to the
concentric pattern seen with non-selected control.
Conditioned Medium Test
The fungus grew well on coverslips bearing PDA or conditioned
medium from non-selected controls. There was no difference in
growth between the two. On the other hand, mycelial growth was
inhibited on coverslips with conditioned medium from both resistant
lines (FIG. 4). Microscopical examination revealed that the hyphal
tips were smaller and many had burst, probably soon after they
started growing onto these coverslips. Additionally, mycelial
growth was uninhibited around these coverslips. This suggests that
the coated coverslips contained anti-fungal compounds that had been
secreted into the culture medium by selected cultures.
Electrophoresis of Extracellular Proteins
Significant differences in extracellular protein profile between
the in vitro selected lines and non-selected controls could be seen
in the SDS-PAGE, both in PEMs and differentiated somatic embryos.
PEMs of both selected lines secreted additional proteins of 8, 22
and 33 kDa (FIG. 5A). Heart stage somatic embryos of non-selected
controls exhibited two proteins of 35 and 36 kDa, while there was
only one protein of 36 kDa in the selected lines (FIG. 5B). In
addition, the 22 and 33 kDa proteins secreted by PEMs of selected
lines were also present during this stage of embryogenesis, but the
8 kDa protein was absent. It is possible that this protein was
present, but ran out of the gel, since shorter electrophoretic runs
could not resolve this region adequately.
Chitinase Activity in Extracellular Proteins
Native PAGE, which can resolve even isozymes of the same size,
indicates that selected lines have multiple chitinases. Two of
these are induced by selection. One isozyme, with the least
mobility, was greatly elevated in the selected lines in comparison
with the control. After SDS-PAGE analysis, a 36 kDa protein
exhibited chitinase activity in both selected lines and the
non-selected controls as revealed by glycol chitin gel assay. At
least a twenty-fold increase in chitinase activity of the 36 kDa
isozyme was seen in the resistant lines as revealed by
densitometric analysis. A 28 kDa protein also showed chitinase
activity in the resistant lines. The results indicate that new
isozymes of chitinases are expressed after selection and that the
secretion of chitinase increases after in vitro selection in
grapevine embryogenic cultures.
Immunological Detection of Chitinase
The 36 kDa protein in the ECP of both resistant lines strongly
reacted with chitinase antiserum. There was no reaction in the ECP
of non-selected controls, though chitinase activity was detected in
the glycol chitin assay. This indicates that the 36 kDa protein
observed in the non-selected control and in the selected lines may
not be the same protein. The 28 kDa peptide that was present in the
selected lines as detected in the glycol chitin assay, did not
react with this antiserum.
Retesting of Somatic Embryos after In Vitro Selection
Mature somatic embryos of non-selected controls grew normally on
germination medium, but they did not germinate on medium containing
40% (v/v) fungal culture filtrate. Most of them turned necrotic
within 4 days after culture. Somatic embryos from both resistant
lines germinated and grew into plants on both media (FIG. 7),
indicating that the acquired resistance is stable and not
epigenetic. More than 50 plants were regenerated from somatic
embryos from each of the resistant lines and established in the
greenhouse. Plant establishment was accomplished at 8 months after
selection and testing of plants occurred when they were 18 months
old.
In Vitro Leaf Bioassay
Leaves from non-selected controls developed black lesions at the
infected sites within three days of spore inoculation. The lesions
spread rapidly and the entire leaf became necrotic within a week.
Leaves from both in vitro selected lines were very slow in
exhibiting the lesions. It took more than 10 days for the lesions
to appear. The lesions did not spread as in the controls, even
after two weeks of incubation, indicating that the resistance
acquired by PEMs during in vitro selection persisted in the
plants.
Testing the Regenerated Plants for Resistance
After removing bags, the leaves of inoculated plants were examined
for disease symptoms. Most of the young leaves from non-selected
control plants were crinkled with spreading lesions. Some leaves
exhibited `shothole symptoms`, characteristic of anthracnose
disease (FIG. 8). Few leaves turned necrotic within this three day
period. There was extensive defoliation among non-selected
controls. Thirty nine out of forty in vitro selected plants from
both resistant lines remained healthy even after several days. Only
one plant tested this way showed mild symptoms of leaf curl; no
lesions were observed, however. Defoliation was very minimal and
often only the older leaves were lost.
Re-Isolation of Fungus from the Infected Plants
Fungal mycelium grew rapidly from symptomatic leaves of control
plants. Mycelial growth was identical to that of the original
control culture. Microscopic observations of conidia confirmed them
to be E. ampelina. Koch's postulate was accomplished using these
conidia to infect grapevine leaves.
Identification of Differentially Expressed Proteins
Extracellular proteins from resistant and control embryogenic
cultures were analyzed to determine if any activation of defense
genes was apparent in the embryogenic cells or somatic embryos
resistant to E. ampelina. Analysis of extracellular proteins (i.e.,
proteins secreted in the liquid culture medium) revealed changes in
protein profiles between the control and resistant embryogenic
cultures. In addition, chitinase was observed to be secreted in
abundance by the resistant embryogenic cultures in comparison with
control cultures. This secretion of chitinase was observed even
eight months after selection. These results demonstrated that the
resistant cultures retained an activity many generations (in terms
of cell divisions) after the selection pressure had been removed;
hence, the E. ampelina resistance was a stable genetic
mutation.
Extraction of proteins in the intercellular fluids was more
difficult than described for other species. Extracted proteins are
preferably separated by electrophoresis within a few hours, since
storing them even at -50.degree. C. leads to loss of proteins. ICWF
extractions were analysed several times in order to confirm the
separation of proteins. Two prominent, differentially expressed,
proteins of 8 and 22 kDa could be identified consistently in the
ICWF of selected lines. While there were two proteins of 1.6 and 22
kDa in the ICWF of RC1 and RC2, a weak 21.6 kDa protein was present
in the ICWF of non-selected control plants (FIG. 5C).
Immunodetection
The 22 kDa protein from both resistant lines reacted with pinto
bean PR 5 antiserum. There was no reaction in the control. This
protein could be detected both at the PEM stage (FIG. 10A) and also
at the heart stage somatic embryo, using the same antiserum,
indicating persistent expression of this protein. At the somatic
embryo stage, however, an additional band of approximately 26 kD
also cross reacted with this antiserum (FIG. 10B) in the resistant
line RC2. There were two bands of 22 and 23 kDa (referred to herein
as the 22 kDa doublet) in the ICWF from plants of both resistant
lines that reacted with the PR-5 antiserum. There was also faint
reaction in the ICWF from non-selected controls (FIG. 10C). Thus
there is a doublet between 22 kDa and 23 kDa that includes two
PR-5-related proteins.
Identification of Differentially Expressed Proteins Using
N-terminal Amino Acid Sequencing
The sequence of the N-terminal 21 amino acids of the 8 kDa protein
was determined by Edman degradation method to be
TVTXGQVASAVGPXISYLQ (SEQ ID NO: 1). Sequence similarity searches
revealed that this protein exhibits a high similarity with
non-specific lipid transfer proteins (nsLTP). Among the nsLTPs that
showed high similarity was a 9 kDa protein identified from
grapevine somatic embryos and identified as LTP P4 (Coutos-Thevenot
et al., Eur. J. Biochem. 217:885 889, 1993). In additon, it also
exhibited 75% similarity with another 9 kDa protein from grapevine
berries (Salzman et al., supra). Thus the 8 kDa protein was
identified as a nsLTP (FIG. 11). Amino acid sequence information
could not be obtained for the 14 kDa protein that was
differentially expressed by heart stage somatic embryos presumably
because the N-terminus of this protein was blocked.
One of the N-terminal amino acid sequences (ATFDILNKXTYTVXA; SEQ ID
NO: 2) of the 22 kDa protein doublet secreted by heart stage
somatic embryos of in vitro selected lines matched that of a
thaumatin/osmotin-like protein (VVTL-1) isolated from grapevine
berries (Tattersal et al., Plant Physiol. 114:759 769, 1997). In
addition, it also exhibited very high sequence similarity with the
N-terminal sequences of several other TLPs. Among these, two
tobacco thaumatin-like proteins, E22 and E2, exhibited 92% sequence
similarity (FIG. 12). The amino fragment from the second protein
(ATFNIQNKGGYTVXA; SEQ ID NO: 3) had homology to grapevine osmotin.
Both proteins from ICWF exhibited high homology with the
corresponding 22 kDa protein doublet secreted by heart stage
somatic embryos. It is evident that the 22 kDa protein doublet is
differentially and constitutively expressed by the selected lines,
predominantly as a secreted protein and could be traced from the
early PEM stage to all the way in regenerated plants.
N-terminal sequence of the 33 kDa protein from heart stage embryos
(ASLADQQANEFTKV; SEQ ID NO: 4) did not reveal any significant
sequence similarity in the database search. A cDNA encoding the 33
kDa protein was cloned as follows. Primers were designed based on
amino terminal and carboxy terminal amino acid sequence information
generated from the 33 kDa protein. Using these, we amplified the
fragment from the genomic DNA and then cloned and sequenced the
fragment. The primer designed based on the carboxy terminal
fragment did not help in amplifying, but a palindromic sequence to
the primer designed based on the N terminal fragment existed at the
3' end of the DNA sequence. The sequences for both the DNA (SEQ ID
NO: 6) and the putative protein (SEQ ID NO: 5) are depicted in FIG.
9.
Pathogen Resistance
The methods of the invention are useful for providing resistance to
other grapevine diseases. Grape plants exhibiting resistance to a
number of different diseases may be generated from embryogenic
cells and embryogenic cell masses that are selected for resistance
to the etiologic agent of a particular disease, a toxin produced by
the agent, or the etiologic agent (or toxin) of another grapevine
disease. For example, embryogenic cells and embryogenic cell masses
may be grown in a liquid suspension culture in the presence of a
filter-sterilized culture filtrate prepared from a pathogen, at a
concentration of culture filtrate that is ideal for in vitro
selection. After four or five cycles of recurrent selection in such
a liquid medium containing culture filtrate, with subculturing
performed every ten days as described above, the surviving cells
are allowed to expand in a liquid medium lacking the culture
filtrate. From these cells, somatic embryogenesis may be performed
to produce cells and plants showing increased resistance to the
powdery mildew disease, as well as to diseases caused by other
fungi and/or bacteria. The filtrate may be the cell supernatant
from the culture. In some cases, it may be preferable to culture
the pathogen in the presence of plant cells, harvest and lyse
and/or homogenize the cells, and then collect the supernatant
following centrifugation. Such a filtrate is particularly useful
when the pathogen is a virus or a bacterium.
The method described herein can be modified to select for cells
that have been transformed with a nucleic acid sequence. Cell
transformation, while a standard technique, does not result in
every cell containing the nucleic acid of interest. It is standard
laboratory practice to include in the transformation nucleic acid
sequence that confers a growth advantage in a specific selection
medium. Thus, only the cells of interest (i.e., the ones that are
transformed) are able to grow or survive in the selection medium.
The proteins described herein (and the nucleic acids encoding them)
can be used as selectable markers in such methods. In this example,
the selection medium includes a pathogen, or a pathogen filtrate or
conditioned medium. Cells that have been transformed with the
nucleic acid sequence encoding the protein that confers pathogen
resistance will survive, while cells that have not been transformed
will die.
It will be understood that a protein that confers resistance to one
pathogen may also confer resistance to additional pathogens. Plants
resistant to anthracnose may be additionally resistant to
additional pathogens. For example, a plant that is resistant to
both anthracnose and black rot (caused by the fungus, Guignardia
bidwellii) may be additionally resistant to Botrytis bunch rot and
blight (caused by the fungus, Botrytis cinerea). The rapid
generation of these resistant grape plants using the methods of the
invention allows for such combination of resistance not just for
fungi, but for other grapevine pathogens (e.g., bacteria and
viruses).
Evaluation of the level of pathogen protection conferred to a plant
by the selection methods described herein is determined according
to conventional methods.
EXAMPLE 5
Grapevine Transformation
The method described herein can be used to produce transformed
plants. Cells can be transformed at any step in the process of
making a somatic embryo-derived. Thus, tissue or cells suitable for
transformation include explanted tissue, embryogenic cells,
embryogenic cell masses, and somatic embryos (including mature
somatic embryos).
Cell cultures produced according to the methods of the invention
may be transformed with DNA comprising a desired transgene, such as
the DNA of SEQ ID NO: 6). Such cells, for example, may be
transformed with genes which confer resistance to pathogens,
diseases, or pests, or any combination thereof. For example, a
number of Bacillus thurigiensis genes which encode proteins that
are toxic to a number of pests are well known and useful in the
methods of the invention. Several standard methods are available
for introduction of a transgene into a plant host, thereby
generating a transgenic plant.
Upon construction of the plant expression vector, several standard
methods are available for introduction of the vector into a plant
host, thereby generating a transgenic plant. These methods include
(1) Agrobacterium-mediated transformation (A. tumefaciens or A.
rhizogenes) (see, e.g., Lichtenstein and Fuller In: Genetic
Engineering, vol 6, P W J Rigby, ed, London, Academic Press, 1987;
and Lichtenstein, C. P., and Draper, J, In: DNA Cloning, Vol II, D.
M. Glover, ed, Oxford, IRI Press, 1985)); (2) the particle delivery
system (see, e.g., Gordon-Kamm et al., Plant Cell 2:603 (1990); or
BioRad Technical Bulletin 1687, supra); (3) microinjection
protocols (see, e.g., Green et al., supra); (4) polyethylene glycol
(PEG) procedures (see, e.g., Draper et al., Plant Cell Physiol.
23:451, 1982; or e.g., Zhang and Wu, Theor. Appl. Genet. 76:835,
1988); (5) liposome-mediated DNA uptake (see, e.g., Freeman et al.,
Plant Cell Physiol. 25:1353, 1984); (6) electroporation protocols
(see, e.g., Gelvin et al., supra; Dekeyser et al., supra; Fromm et
al., Nature 319:791, 1986; Sheen Plant Cell 2:1027, 1990; or Jang
and Sheen Plant Cell 6:1665, 1994); and (7) the vortexing method
(see, e.g., Kindle supra). The method of transformation is not
critical to the invention. Any method which provides for efficient
transformation may be employed. As newer methods are available to
transform crops or other host cells, they may be directly
applied.
The following is an example outlining one particular technique, an
Agrobacterium-mediated plant transformation. By this technique, the
general process for manipulating genes to be transferred into the
genome of plant cells is carried out in two phases. First, cloning
and DNA modification steps are carried out in E. coli, and the
plasmid containing the gene construct of interest is transferred by
conjugation or electroporation into Agrobacterium. Second, the
resulting Agrobacterium strain is used to transform plant cells.
Thus, for the generalized plant expression vector, the plasmid
contains an origin of replication that allows it to replicate in
Agrobacterium and a high copy number origin of replication
functional in E. coli. This permits facile production and testing
of transgenes in E. coli prior to transfer to Agrobacterium for
subsequent introduction into plants. Resistance genes can be
carried on the vector, one for selection in bacteria, for example,
streptomycin, and another that will function in plants, for
example, a gene encoding kanamycin resistance or herbicide
resistance. Also present on the vector are restriction endonuclease
sites for the addition of one or more transgenes and directional
T-DNA border sequences which, when recognized by the transfer
functions of Agrobacterium, delimit the DNA region that will be
transferred to the plant.
In another example, plant cells may be transformed by shooting into
the cell tungsten microprojectiles on which cloned DNA is
precipitated. In the Biolistic Apparatus (Bio-Rad) used for the
shooting, a gunpowder charge (22 caliber Power Piston Tool Charge)
or an air-driven blast drives a plastic macroprojectile through a
gun barrel. An aliquot of a suspension of tungsten particles on
which DNA has been precipitated is placed on the front of the
plastic macroprojectile. The latter is fired at an acrylic stopping
plate that has a hole through it that is too small for the
macroprojectile to pass through. As a result, the plastic
macroprojectile smashes against the stopping plate, and the
tungsten microprojectiles continue toward their target through the
hole in the plate. For the instant invention the target can be any
plant cell, tissue, seed, or embryo. The DNA introduced into the
cell on the microprojectiles becomes integrated into either the
nucleus or the chloroplast.
In general, transfer and expression of transgenes in plant cells
are now routine practices to those skilled in the art, and have
become major tools to carry out gene expression studies in plants
and to produce improved plant varieties of agricultural or
commercial interest.
While the expression of one of the proteins of the invention is
likely to confer on a plant increased disease resistance, it may be
preferable to express two, three, or even all four proteins in a
plant to achieve maximal pathogen resistance. This can be achieved
either by the selection method described herein, or by producing a
plant having transgenes encoding the four sequences.
EXAMPLE 6
Generation of Antibodies, Nucleic Acids, and Proteins
Using standard techniques, such as those described above, one in
the art can identify full-length proteins and nucleic acids from
any variety of grape plant. For example, an amino terminal peptide
fragment can be used to generate a degenerate nucleic acid probe
for PCR, Southern blotting, or colony hybridization. Using a
nucleic acid sequence, one can identify orthologues in other
variety of plants or in plants other than grape plants. The
proteins or polypeptides of the invention can be used to raise
antibodies or binding portions thereof or probes. The antibodies
can be monoclonal or polyclonal. A description of the theoretical
basis and practical methodology of fusing such cells is set forth
in Kohler and Milstein, Nature, 256:495, 1975), and Milstein and
Kohler, Eur. J. Immunol., 6:511, 1976), hereby incorporated by
reference. Procedures for raising polyclonal antibodies are also
well known to the skilled artisan. This and other procedures for
raising polyclonal antibodies are disclosed in Harlow et. al.,
editors, Antibodies: A Laboratory Manual (1988), which is hereby
incorporated by reference.
In addition to utilizing whole antibodies, binding portions of such
antibodies can be used. Such binding portions include Fab
fragments, F(ab').sub.2 fragments, and Fv fragments. These antibody
fragments can be made by conventional procedures, such as
proteolytic fragmentation procedures, as described in Goding,
Monoclonal Antibodies: Principles and Practice, New York: Academic
Press, pp. 98 118 (1983), hereby incorporated by reference.
The present invention also relates to probes found either in nature
or prepared synthetically by recombinant DNA procedures or other
biological procedures. Suitable probes are molecules which bind to
the proteins of the present invention. Such probes can be, for
example, proteins, peptides, lectins, or nucleic acid probes.
Antibodies raised against the proteins or polypeptides of the
present invention or binding portions of these antibodies can be
utilized in a method for selection of plants having increased
resistance to a plant pathogen. A variety of assay systems can be
employed, such as enzyme-linked immunosorbent assays,
radioimmunoassays, gel diffusion precipitin reaction assays,
immunodiffusion assays, agglutination assays, fluorescent
immunoassays, protein A immunoassays, or immunoelectrophoresis
assays.
The sequences of the present invention can also be used to identify
proteins that are substantially identical to those described
herein. By "substantially identical" is meant a protein or nucleic
acid exhibits at least 70%, preferably 80%, and most preferably
90%, 95%, or even 98% identity to a reference amino acid sequence
or nucleic acid sequence. For proteins, the length of comparison
sequences will generally be at least 15 amino acids, preferably at
least 20 amino acids, more preferably at least 25 amino acids, and
most preferably 35 amino acids or greater. For nucleic acids, the
length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides or
greater.
Sequence identity, at the amino acid levels, is typically measured
using sequence analysis software (for example, Sequence Analyis
Software Package of the Genetics Computer Group, Univerity of
Wisconsin Biotechnology Center, 1710 University Avenue, Madison,
Wis. 53705, BLAST, or PILEUP/PRETTYBOX prgrams). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications.
The present invention also includes nucleic acids that selectively
hybridize to the DNA sequence of the present invention.
Hybridization may involve Southern analysis (Southern Blotting), a
method by which the presence of DNA sequences in a target nucleic
acid mixture are identified by hybridization to a labeled
oligonucleotide or DNA fragment probe. Southern analysis typically
involves electrophoretic separation of DNA digests on agarose gels,
denaturation of the DNA after electrophoretic separation, and
transfer of the DNA to nitrocellulose, nylon, or another suitable
membrane support for analysis with a radiolabeled, biotinylated, or
enzyme-labeled probe as described in Sambrook et al., (1989)
Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.
Hybridization often includes the use of a probe. It is generally
preferred that a probe of at least 20 nucleotides in length be
used, preferably at least 50 nucleotides, more preferably at least
about 100 nucleotides.
A nucleic acid can hybridize under moderate stringency conditions
or high stringency conditions to a nucleic acid disclosed herein.
High stringency conditions are used to identify nucleic acids that
have a high degree of homology or sequence identity to the probe.
High stringency conditions can include the use of a denaturing
agent such as formamide during hybridization, e.g., 50% formamide
with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, and 75 mM sodium citrate at 42.degree. C. Another
example is the use of 50% formamide, 5.times.SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times. Denhart's solution, sonicated salmon
sperm DNA (50 ug/mL) 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC and
0.1% SDS. Alternatively, low ionic strength washes and high
temperature can be employed for washing.
Moderate stringency conditions are hybridization conditions used to
identify nucleic acids that have less homology or identity to the
probe than do nucleic acids under high stringency. All of these
techniques are well known to the artisan skilled in molecular
biology.
Materials and Methods
In Vitro Selection, Culture Establishment and Plant
Regeneration
Suspension cultures, somatic embryogenesis and plant regeneration
of `Chardonnay` (Clone 02Ch; Stimson Lane Wineries, Prosser, Wash.)
consisting of actively growing PEMs were established as follows.
Log phase cultures were sieved using a 960 .mu.M sieve to generate
a synchronized culture. Approximately 1.0 g of PEMs were subjected
to recurrent selection in suspension culture with a modified
culture medium containing 40% (v/v) fungal culture filtrate. The
liquid medium was prepared and cooled to room temperature and the
culture filtrate was added after filter sterilization to eliminate
any loss of filtrate activity due to autoclaving. The culture
filtrate was obtained by growing a virulent strain of E. ampelina
spores for 3 weeks in Czapek-Dox broth. Cell free extract was
collected and stored at -20.degree. C. until further use. The pH of
the culture was adjusted to 5.8 before adding to the medium.
Selection was carried out for four or five cycles, each cycle
lasting for 10 days. At the end of 4.sup.th and 5.sup.th cycles,
putative resistant cultures were proliferated in regular suspension
culture medium and established as `resistant culture 1` (RC1) and
`resistant culture 2` (RC2), respectively. Somatic embryogenesis
was achieved by culturing the selected PEMs in auxin-free
suspension culture medium and the resulting somatic embryos were
germinated in solid medium. Regenerated plants were acclimatized in
potting mixture and established in a greenhouse. A set of control,
non-selected PEMs were cultured in a similar way and plants
regenerated from these non-selected cultures served as control for
rest of the experiments.
Dual Cultures
PEMs of resistant and unchallenged controls, both of which were
maintained in suspension culture for more than 20 weeks after
selection, were used for dual culture. PEMs were collected on a
sterile filter paper and approximately 1.0 g of PEMs were cultured
on semisolid medium at opposite sides of a 100.times.15 mm petri
dish. The medium had the same components as liquid medium, but was
solidified with TC agar at 7.0 g/l. The cultures were sealed with
Parafilm.TM. and incubated in darkness at 25.+-.2.degree. C. After
5 weeks, a mycelial plug (5 mm in diameter) from an actively
growing fungal culture was placed at the center of the plates.
Cultures were tested against two different fungi, E. ampelina
(against which the PEMs were selected) and Fusarium oxysporium (a
root pathogen isolated from watermelon). After inoculating mycelial
plugs the cultures were sealed and incubated at 25.+-.2.degree. C.
at 16 h photoperiod. There were 5 petri plates for each fungus and
the experiment was repeated twice. Mycelial growth on the plates
was measured daily and photographed after 10 days of culture.
Conditioned Medium Assay
Spent liquid medium was collected from a resistant line and
unchallenged control and centrifuged at 2500 rpm for 10 min to
remove cellular debris. After filter-sterilization, the supernatant
was diluted with an equal volume of warm, 1.5 N (58.5 gl.sup.-1)
potato dextose agar (PDA) medium to give a final concentration of
0.75 N (29.25 gl.sup.-1). Sterile glass slide coverslips were
soaked in the molten medium rapidly (before the medium solidified)
and placed on 0.75 N PDA plates. Three coverslips were placed on
each plate. Coverslips soaked in 0.75 N PDA and plated as before
served as an additional control. After cooling the plates
overnight, a mycelial plug from E. ampelina was placed at the
center of the plate and incubated at 25.+-.2.degree. C. Mycelial
growth on the coverslips containing the conditioned medium was
evaluated daily and photographed after seven days of culture.
Extraction of ECPs
Spent medium was collected in sterile flasks during subculture and
filtered through a double layer Kimwipe.TM. to eliminate any
cellular debris. ECP was precipitated from the filtered medium by
adding three volumes of ice cold, 95% ethanol and kept overnight at
0.degree. C. Proteins were pelleted by centrifugation, concentrated
in a vacuum concentrator and resuspended in sterile distilled
water. Protein quantitation was done by the Bradford protein assay,
using bovine serum albumin as a standard. Protein samples were
stored at -20.degree. C. until further use.
Extraction of ICWF and Protein Concentration
Fully expanded, flaccid leaves were collected from greenhouse-grown
plants early in the morning. The leaves were washed thoroughly with
distilled water and blot-dried. Lamina were cut into 2 cm wide
strips and vacuum infiltrated for 15 min in a buffer containing 100
mM Tris-HCl, 2.0 mM CaCl.sub.2, 10 mM EDTA, 50 mM P-mercaptoethanol
and 0.5 M sucrose, at the rate of 10 ml/g of leaf tissue. After
infiltration, the leaf strips were gently blotted and rolled into
0.5 ml microfuge tubes (without caps) with a 0.2 mm dia hole at the
bottom. Only 2 3 strips were loaded in each microfuge tube. These
tubes were then loaded onto a 1.5 ml centrifuge tubes. The set-up
was spun at 7500 rpm for 15 min at room temperature. ICWF collects
as a dense drop in the 1.5 ml centrifuge tubes. To concentrate the
proteins, ICWF was diluted with 4 volumes of distilled water and
the proteins were precipitated with 3 volumes of ice-cold, 95%
ethanol overnight at 0.degree. C. Proteins were pelleted by
centrifugation, concentrated in a vacuum concentrater and
re-suspended in sterile distilled water.
Electrophoresis of Proteins
SDS-PAGE was carried out using 1 mm thick mini gels. Protein
samples were diluted with equal volume of SDS-PAGE buffer (Sigma,
St. Louis, Mo.) and the diluted samples were heated in a boiling
water bath for 5 min and cooled. Samples were spun at 10,000 rpm
for 5 min at room temperature to remove any insoluble particles.
Total protein of 10 .mu.g was loaded onto each lane and
electrophoresed for approximately 80 min at 200 V. The gels were
then either silver-stained using SilverSnap.TM. (Pierce, Rockford.
Ill.) or stained with colloidal Coomassie Blue (Sigma, St. Louis
Mo.) and photographed using a Kodak DC 120 digital camera.
Chitinase activity was analyzed as follows. After native PAGE, the
gels were rinsed in 150 .mu.M sodium acetate (pH 5.0) for 15 min.
The gels were placed on a clean glass plate and overlaid with a
7.5% gel containing 0.01% (v/v) glycol chitin. After removing air
bubbles, the gel sandwich was incubated at 37.degree. C. under
moist conditions. The overlay gels were removed and stained with
0.01% fluorescent brightener (Calcoflour white M2R) in Tris-HCl
buffer (pH 8.9) for 10 min and rinsed thoroughly in distilled water
overnight. Chitinase activity derived from various chitinase
isozymes was visible as dark (lytic) bands in the overlay gels.
Running gels in SDS-PAGE were incorporated with 0.02% glycol chitin
while casting the gels. After electrophoresis, the gels were
incubated in 200 mM sodium acetate solution at pH 5.0 containing 1%
of Triton-X 100 for 4 h at 37.degree. C. After incubation, the gels
were washed 3 times with distilled water, stained with 0.01% (v/v)
fluorescent brightener in 500 mM Tris-HCl (pH 8.9) for 10 min and
destained overnight in distilled water. Chitinase isozymes were
identified as lytic bands on a UV-transilluminator and photographed
using a Kodak DC 120 digital camera with orange filter.
Immunodetection of Chitinase
SDS-PAGE was carried out as described above and the proteins were
transferred to a PVDF membrane (Bio-Rad, Almeda, Calif.) in a mini
transblot gel transfer cell. Following transfer of proteins, the
membrane was probed with an antiserum raised against a barley seed
chitinase at 1:1000 dilution. The antigen-antibody complex was
detected by a goat-anti rabbit horseradish peroxidase
(Bio-Rad).
Re-Testing the In Vitro Selected Cultures for Resistance to Culture
Filtrate
Mature somatic embryos from both selected cultures and non-selected
control were germinated on a solid germination medium containing
40% (v/v) fungal culture filtrate. There were five plates per
treatment, each containing 15 embryos. After culturing, the plates
were incubated in the dark. Three weeks after incubation, embryos
that germinated were counted as being resistant. A similar set of
embryos were germinated in a medium without culture filtrate as an
additional control.
Plant Regeneration and Establishment in Greenhouse
Plants regenerated from somatic embryos were transferred to starter
plugs containing sterile commercial potting mixture and kept under
16 h photoperiod for in vivo acclimitization. After approximately
one month, soil-acclimatized plants were transferred to the
greenhouse. Well-established and vigorously growing plants,
approximately 18 months after regeneration, were used for further
studies.
In Vitro Leaf Bioassay for Anthracnose Resistance
Fully expanded green, young leaves, approximately 6 cm wide were
collected from 10 different plants in each of two selected lines
and the non-selected control. These leaves were inoculated with 100
.mu.l of a spore suspension containing 1.times.10.sup.6 spores per
ml. There were three inoculations on each leaf in the inter-venal
region. Immediately after inoculation, the leaves were incubated
under humid conditions in moist chambers at 25+2.degree. C. and 16
h photoperiod. After one week, the leaves were evaluated for
anthracnose symptoms. The assay was repeated twice.
Test for Anthracnose Resistance in Selected Plants
Eighteen month old, greenhouse grown in vitro selected plants and
non-selected controls regenerated from somatic embryos were used in
this study. Clones from the original `Chardonnay` (`02Ch`), from
which the cultures were initiated, were also used as an additional
control. Plants that were actively growing with young leaves were
chosen for this test. The plants were sprayed with a spore
suspension containing 1.times.10.sup.6 spores per ml on both sides
of the leaves until runoff. They were then individually covered
with a polythene bag carefully so that leaves did not touch the bag
which was sealed around the pot. These plants were incubated in the
growth room, at 25+/-2.degree. C. and 16 h photoperiod. After 72 h
of incubation, the bags were carefully removed and the plants were
observed for disease symptoms. Plants exhibiting crinkling of leaf
lamina or typical shot hole symptoms were scored as susceptible.
The experiment was repeated twice using different sets of plants
from each selected line and control. For each test, there were at
least 20 plants from each selected line and 6 plants from the
control.
Recovery of Pathogen after Infection
Leaves that showed anthracnose symptoms were removed and washed
well with distilled water. They were air dried under the laminar
flow hood for two days. Pieces of lamina and midrib from these air
dried leaves were then cultured in PDA. A small plug of mycelium
from the original culture that was used to infect the leaves was
also cultured, for comparison. The cultures were incubated at 16 h
photoperiod and 25.+-.2.degree. C.
Immunodetection of Proteins
After SDS-PAGE, proteins were transferred to ImmunoBlot.TM. PVDF
membrane (Bio-Rad, Almeda, Calif.) in a mini trans-blotter
according to manufacturer's instructions. Transfer was carried out
under high intensity electric field (100 V) for 2 hr. The membrane
was washed thoroughly in washing buffer and rinsed thrice in
distilled, deionized water for 10 min each, then blocked overnight
in a 3% bovine serum albumin solution at room temperature. After
another cycle of washing and rinsing, proteins were probed with PR
5 antiserum raised against pinto bean thaumatin-like protein
(provided by Dr. O. P. Sehgal, University of Missouri, Columbia,
Mo.) at a dilution of 1:500 for 2 hr at room temperature with
gentle shaking. Color development was carried out using Opti-4Cn
kit (Bio-rad, Almeda, Calif.), according to manufacturer's
instructions.
N-Terminal Amino Acid Sequencing
For N-terminal amino acid sequencing, proteins were transferred to
an ImmunoBlot PVDF membrane using a buffer lacking glycine. After
transfer, proteins were stained with Coomassie blue and appropriate
bands were identified based on their molecular weight and cut out
using a sterile scalpel. Amino-terminal amino acid sequence
determination was accomplished by the automated Edman degradation
method, in the Protein Chemistry Core Laboratory, University of
Florida, Gainesville, using a protein sequencer, Model 494HT
(Applied Biosystems, Foster City, Calif.). Phenylthiohydantoin
amino acid derivatives were automatically detected by a 120A
analyzer used in conjunction with the sequencer.
Other Embodiments
All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication was specifically and individually indicated to be
incorporated by reference.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of
further modifications. This application is intended to cover any
variations, uses, or adaptations following, in general, the
principles of the invention and including such departures from the
present disclosure within known or customary practice within the
art to which the invention pertains and may be applied to the
essential features hereinbefore set forth.
SEQUENCE LISTINGS
1
6119PRTVitis viniferaVARIANT(1)...(19)Xaa = Any Amino Acid 1Thr Val
Thr Xaa Gly Gln Val Ala Ser Ala Val Gly Pro Xaa Ile Ser 1 5 10
15Tyr Leu Gln215PRTVitis viniferaVARIANT(1)...(15)Xaa = Any Amino
Acid 2Ala Thr Phe Asp Ile Leu Asn Lys Xaa Thr Tyr Thr Val Xaa Ala 1
5 10 15315PRTVitis viniferaVARIANT(1)...(15)Xaa = Any Amino Acid
3Ala Thr Phe Asn Ile Gln Asn Lys Gly Gly Tyr Thr Val Xaa Ala 1 5 10
15414PRTVitis vinifera 4Ala Ser Leu Ala Asp Gln Gln Ala Asn Glu Phe
Thr Lys Val 1 5 105251PRTVitis vinifera 5Ala Asn Glu Phe Thr Asn
Leu Leu Tyr Cys Ile Gln Lys Arg Lys Lys 1 5 10 15Lys Tyr Val Ile
Phe Gly Val Cys Asp Val Tyr Gly Ile His Gln Gly 20 25 30Gly Ile Ile
Leu Gly Pro Ser Gly Leu Gly Lys Ser Pro Ala Phe Ser 35 40 45Lys Trp
Val Phe Pro Glu Ser Ser Ile Tyr Phe Ser Gln Thr Val Ala 50 55 60Leu
Phe Gly Cys Met Ile Phe Met Phe Leu Val Gly Val Lys Met Asp65 70 75
80Thr His Leu Met Arg Lys Ser Gly Arg Arg Gly Val Val Ile Gly Phe
85 90 95Cys Asn Phe Phe Leu Pro Leu Ile Ile Val Val Gly Leu Ala His
Asn 100 105 110Leu Arg Lys Thr Lys Thr Leu Gly His Asn Ile Ser Asn
Ser Ile Tyr 115 120 125Cys Val Ala Thr Leu Met Ser Met Ser Ser Ser
His Val Ile Thr Cys 130 135 140Leu Leu Thr Asp Ile Lys Ile Leu Asn
Ser Glu Leu Gly Arg Leu Ala145 150 155 160Leu Ser Ser Ser Met Ile
Ser Gly Leu Cys Ser Trp Thr Leu Ala Leu 165 170 175Gly Ser Tyr Val
Ile Phe Gln Gly Ser Thr Gly Gln Tyr Glu Ser Met 180 185 190Leu Ala
Leu Ser Leu Ser Phe Ile Ile Leu Val Leu Ile Ile Val Tyr 195 200
205Ile Leu Arg Pro Ile Met Asp Trp Met Val Glu Gln Thr Ala Glu Gly
210 215 220Lys Pro Ile Lys Glu Ser Tyr Val Phe Ser Ile Phe Val Met
Ile Leu225 230 235 240Gly Ser Ala Phe Leu Gly Glu Leu Ile Gly Leu
245 2506777DNAVitis vinifera 6agaattccaa caggccaatg agttcaccaa
tttactgtac tgcatccaaa agaggaaaaa 60gaagtatgta atatttggtg tgtgtgatgt
ttatggtatt catcagggag gtattatcct 120gggaccgtcg ggtttaggaa
aatctccagc attctccaaa tgggttttcc cagagagcag 180catttatttc
agccaaaccg tcgccttatt tgggtgcatg atctttatgt tcctagttgg
240agtgaaaatg gatacacatc tgatgaggaa gtcaggaagg agaggagtag
tcataggctt 300ctgcaacttc ttcttgccat tgataattgt ggttggcttg
gctcacaatc tcagaaaaac 360taagaccttg ggccacaata taagcaattc
tatttactgt gtagcaacac tgatgagcat 420gagttcctcc catgtcatta
cttgccttct aactgatatc aagatcctca actccgagct 480gggaaggtta
gccctatcct catctatgat aagtggcctg tgcagttgga ccctggcatt
540gggctcatat gtaatatttc aaggctcaac tggtcagtat gaaagcatgc
tagcattatc 600cttgtcattt atcatcttgg tgcttatcat tgtatacatt
ctgcggccta ttatggattg 660gatggttgaa cagactgctg aaggaaaacc
aatcaaggag agctatgtct ttagcatctt 720tgtgatgatc ttagggagtg
ccttccttgg tgaactcatt ggcctgttgg aattctt 777
* * * * *